What is Primer ?

<div dir="ltr" style="text-align: left;" trbidi="on"> <h1 class="firstHeading" id="firstHeading" lang="en" style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 1px; font-family: 'Linux Libertine', Georgia, Times, serif; font-size: 1.8em; font-weight: normal; line-height: 1.3; margin: 0px 0px 0.25em; overflow: hidden; padding: 0px;"> <span dir="auto">Primer (molecular biology)</span></h1> <div class="mw-body-content" id="bodyContent" style="color: #252525; font-family: sans-serif; font-size: 0.875em; line-height: 1.6; position: relative;"> <div id="siteSub" style="display: inline; font-size: 13px;"> From Wikipedia, the free encyclopedia</div> <div id="contentSub" style="color: #545454; font-size: 12px; line-height: 1.2em; margin: 0px 0px 1.4em 1em; width: auto;"> </div> <div class="mw-jump" id="jump-to-nav" style="-webkit-user-select: none; height: 0px; margin-bottom: 1.4em; margin-top: -1.4em; overflow: hidden; zoom: 1;"> </div> <div class="mw-content-ltr" dir="ltr" id="mw-content-text" lang="en" style="direction: ltr;"> <div class="thumb tright" style="clear: right; float: right; margin: 0.5em 0px 1.3em 1.4em; width: auto;"> <div class="thumbinner" style="background-color: #f9f9f9; border: 1px solid rgb(204, 204, 204); font-size: 13px; min-width: 100px; overflow: hidden; padding: 3px !important; text-align: center; width: 302px;"> <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"><img alt="" class="thumbimage" data-file-height="336" data-file-width="691" height="146" src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/8f/DNA_replication_en.svg/300px-DNA_replication_en.svg.png" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/8/8f/DNA_replication_en.svg/450px-DNA_replication_en.svg.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/8/8f/DNA_replication_en.svg/600px-DNA_replication_en.svg.png 2x" style="background-color: white; border: 1px solid rgb(204, 204, 204); vertical-align: middle;" width="300" /></span></span><div class="thumbcaption" style="border: none; font-size: 12px; line-height: 1.4em; padding: 3px !important; text-align: left;"> <div class="magnify" style="background: none !important; border: none !important; float: right; margin-left: 3px;"> <span style="color: #0b0080;"><span style="background-attachment: initial !important; background-clip: initial !important; background-origin: initial !important; background-position: initial !important; background-repeat: initial !important; background-size: initial !important;"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf11/skins/common/images/magnify-clip.png" style="background: none !important; border: none !important; display: block; vertical-align: middle;" width="15" /></span></span></div> The DNA replication fork. RNA primer labeled at top.</div> </div> </div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> A <b>primer</b> is a strand of <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">nucleic acid</span></span> that serves as a starting point for <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA synthesis</span></span>. It is required for DNA replication because the <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">enzymes</span></span> that catalyze this process, <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA polymerases</span></span>, can only add new <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">nucleotides</span></span> to an existing strand of DNA. The polymerase starts replication at the <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">3'-end</span></span> of the primer, and copies the <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">opposite strand</span></span>.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">RNA</span></span> (which can be made <i style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; color: #0b0080; text-decoration: none;">de novo</i><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">).</span></span></div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> Many of the laboratory techniques of <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">biochemistry</span></span> and <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">molecular biology</span></span> that involve DNA polymerase, such as <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA sequencing</span></span> and the <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">polymerase chain reaction</span></span> (PCR), require DNA primers. These primers are usually short, <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">chemically synthesized</span></span> <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">oligonucleotides</span></span>, with a length of about twenty bases. They are <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">hybridized</span></span> to a target DNA, which is then copied by the polymerase.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> </div> <div class="toc" id="toc" style="background-color: #f9f9f9; border: 1px solid rgb(170, 170, 170); display: table; font-size: 13px; padding: 7px; zoom: 1;"> <div id="toctitle" style="direction: ltr; text-align: center;"> <h2 style="background: none; border: none; color: black; display: inline; font-size: 13px; line-height: 1.3; margin: 1em 0px 0.25em; overflow: hidden; padding: 0px;"> Contents</h2>  <span class="toctoggle" style="-webkit-user-select: none;"> [<span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">hide</span></span>] </span></div> <ul style="line-height: 1.6; list-style-image: none; list-style-type: none; margin: 0.3em 0px; padding: 0px;"> <li class="toclevel-1 tocsection-1" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">1</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">Mechanism <i>in vivo</i></span><ul style="line-height: 1.6; list-style-image: none; list-style-type: none; margin: 0px 0px 0px 2em; padding: 0px;"> <li class="toclevel-2 tocsection-2" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">1.1</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">Primer removal</span></li> </ul> </li> <li class="toclevel-1 tocsection-3" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">2</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">Uses of synthetic primers</span><ul style="line-height: 1.6; list-style-image: none; list-style-type: none; margin: 0px 0px 0px 2em; padding: 0px;"> <li class="toclevel-2 tocsection-4" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">2.1</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">PCR primer design</span></li> <li class="toclevel-2 tocsection-5" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">2.2</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">Degenerate primers</span></li> </ul> </li> <li class="toclevel-1 tocsection-6" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">3</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">See also</span></li> <li class="toclevel-1 tocsection-7" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">4</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">References</span></li> <li class="toclevel-1 tocsection-8" style="margin-bottom: 0.1em;"><span class="tocnumber" style="background: none; color: #0b0080; text-decoration: none;">5</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"> </span></span><span class="toctext">External links</span></li> </ul> </div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> </div> <h2 style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 1px; color: black; font-family: 'Linux Libertine', Georgia, Times, serif; font-weight: normal; line-height: 1.3; margin: 1em 0px 0.25em; overflow: hidden; padding: 0px;"> <span class="mw-headline" id="Mechanism_in_vivo">Mechanism <i>in vivo</i></span><span class="mw-editsection" style="-webkit-user-select: none; display: inline-block; font-family: sans-serif; font-size: x-small; line-height: 1em; margin-left: 1em; unicode-bidi: -webkit-isolate; vertical-align: baseline; white-space: nowrap;"><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">[</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">edit</span></span><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">]</span></span></h2> <div class="hatnote" style="font-style: italic; margin-bottom: 0.5em; padding-left: 1.6em;"> Further information: <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA replication § The replication fork</span></span></div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> The leading strand of DNA is that strand of the DNA double helix that is orientated in a 5' to 3' manner. Therefore, its complement must be synthesized in a 3'→5' manner. Because <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA polymerase III</span></span> cannot synthesize in the 3'→5' direction, the lagging strand is synthesized in short segments known as <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Okazaki fragments</span></span>. Along the lagging strand's template, <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">primase</span></span> builds RNA primers in short bursts. DNA polymerases are then able to use the free 3'-<span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">OH</span></span> groups on the RNA primers to synthesize DNA in the 5'→3' direction.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> The RNA fragments are then removed by <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA polymerase I</span></span> for prokaryotes or <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA polymerase δ</span></span> for eukaryotes (different mechanisms are used in <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">eukaryotes</span></span> and <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">prokaryotes</span></span>) and new <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">deoxyribonucleotides</span></span> are added to fill the gaps where the RNA was present. <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA ligase</span></span> then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand.</div> <h3 style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-style: none; color: black; line-height: 1.6; margin: 0.3em 0px 0px; overflow: hidden; padding-bottom: 0px; padding-top: 0.5em;"> <span class="mw-headline" id="Primer_removal">Primer removal</span><span class="mw-editsection" style="-webkit-user-select: none; display: inline-block; font-size: x-small; font-weight: normal; line-height: 1em; margin-left: 1em; unicode-bidi: -webkit-isolate; vertical-align: baseline; white-space: nowrap;"><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">[</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">edit</span></span><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">]</span></span></h3> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> In eukaryotic primer removal, DNA polymerase δ extends the Okazaki fragment in <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">5' to 3'</span></span> direction, and when it encounters the RNA primer from the previous Okazaki fragment, it displaces the 5′ end of the primer into a single-stranded RNA flap, which is removed by nuclease cleavage. Cleavage of the RNA flaps involves either <span style="color: #a55858;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">endonuclease 1</span></span> (FEN1) cleavage of short flaps, or coating of long flaps by the single-stranded DNA binding protein <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">replication protein A</span></span> (RPA) and sequential cleavage by <span style="color: #a55858;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Dna2 nuclease</span></span> and FEN1.<sup class="reference" id="cite_ref-1" style="line-height: 1; unicode-bidi: -webkit-isolate;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; white-space: nowrap;">[</span></span>1]</sup></div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> This mechanism is a potential explanation to how <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">HIV</span></span> virus can transform its genome into double stranded DNA from the RNA-DNA formed after <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">reverse transcription</span></span> of its RNA. However, the HIV-encoded <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">reverse transcriptase</span></span> has own ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">sense</span></span> cDNA strand into an <i>antisense</i> DNA to form a double-stranded DNA intermediate.<sup class="reference" id="cite_ref-2" style="line-height: 1; unicode-bidi: -webkit-isolate;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; white-space: nowrap;">[</span></span>2]</sup></div> <h2 style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 1px; color: black; font-family: 'Linux Libertine', Georgia, Times, serif; font-weight: normal; line-height: 1.3; margin: 1em 0px 0.25em; overflow: hidden; padding: 0px;"> <span class="mw-headline" id="Uses_of_synthetic_primers">Uses of synthetic primers</span><span class="mw-editsection" style="-webkit-user-select: none; display: inline-block; font-family: sans-serif; font-size: x-small; line-height: 1em; margin-left: 1em; unicode-bidi: -webkit-isolate; vertical-align: baseline; white-space: nowrap;"><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">[</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">edit</span></span><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">]</span></span></h2> <div class="thumb tright" style="clear: right; float: right; margin: 0.5em 0px 1.3em 1.4em; width: auto;"> <div class="thumbinner" style="background-color: #f9f9f9; border: 1px solid rgb(204, 204, 204); font-size: 13px; min-width: 100px; overflow: hidden; padding: 3px !important; text-align: center; width: 222px;"> <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"><img alt="" class="thumbimage" data-file-height="536" data-file-width="1028" height="115" src="http://upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_RevComp.svg/220px-Primers_RevComp.svg.png" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_RevComp.svg/330px-Primers_RevComp.svg.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_RevComp.svg/440px-Primers_RevComp.svg.png 2x" style="background-color: white; border: 1px solid rgb(204, 204, 204); vertical-align: middle;" width="220" /></span></span><div class="thumbcaption" style="border: none; font-size: 12px; line-height: 1.4em; padding: 3px !important; text-align: left;"> <div class="magnify" style="background: none !important; border: none !important; float: right; margin-left: 3px;"> <span style="color: #0b0080;"><span style="background-attachment: initial !important; background-clip: initial !important; background-origin: initial !important; background-position: initial !important; background-repeat: initial !important; background-size: initial !important;"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf11/skins/common/images/magnify-clip.png" style="background: none !important; border: none !important; display: block; vertical-align: middle;" width="15" /></span></span></div> Diagrammatic representation of the forward and reverse primers for a standard <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">PCR</span></span></div> </div> </div> <div class="hatnote" style="font-style: italic; margin-bottom: 0.5em; padding-left: 1.6em;"> For the organic chemistry involved, see <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Oligonucleotide synthesis</span></span>. For possible methods involving primers, see <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Nucleic acid methods</span></span>.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">DNA sequencing</span></span> is used to determine the nucleotides in a DNA strand. The <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Sanger chain termination method</span></span> of sequencing uses a primer to start the chain reaction.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> In <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">PCR</span></span>, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 (usually 18–24<sup class="reference" id="cite_ref-3" style="line-height: 1; unicode-bidi: -webkit-isolate;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; white-space: nowrap;">[</span></span>3]</sup>) nucleotides, and they need to match the beginning and the end of the DNA fragment to be amplified. They direct replication towards each other – the extension of one primer by polymerase then becomes the template for the other, leading to an exponential increase in the target segment.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> It is worth noting that primers are not always for DNA synthesis, but can in fact be used by viral polymerases, e.g. influenza, for RNA synthesis.</div> <h3 style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-style: none; color: black; line-height: 1.6; margin: 0.3em 0px 0px; overflow: hidden; padding-bottom: 0px; padding-top: 0.5em;"> <span class="mw-headline" id="PCR_primer_design">PCR primer design</span><span class="mw-editsection" style="-webkit-user-select: none; display: inline-block; font-size: x-small; font-weight: normal; line-height: 1em; margin-left: 1em; unicode-bidi: -webkit-isolate; vertical-align: baseline; white-space: nowrap;"><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">[</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">edit</span></span><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">]</span></span></h3> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> Pairs of primers should have similar melting temperatures since annealing in a PCR occurs for both simultaneously. A primer with a <i>T</i><sub style="line-height: 1;">m</sub> (melting temperature) significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while <i>T</i><sub style="line-height: 1;">m</sub> significantly lower than the annealing temperature may fail to anneal and extend at all.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby. A commonly used method is<span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">BLAST</span></span> search whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched. The free <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">NCBI</span></span> tool Primer-BLAST integrates primer design tool and BLAST search into one application,<sup class="reference" id="cite_ref-4" style="line-height: 1; unicode-bidi: -webkit-isolate;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; white-space: nowrap;">[</span></span>4]</sup> so does commercial software product such as ePrime, <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Beacon Designer</span></span>. Computer simulations of theoretical PCR results (<span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Electronic PCR</span></span>) may be performed to assist in primer design.<sup class="reference" id="cite_ref-ePCR_5-0" style="line-height: 1; unicode-bidi: -webkit-isolate;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; white-space: nowrap;">[</span></span>5]</sup></div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> Many online tools are freely available for primer design, some of which focus on specific applications of PCR. The popular tools <span style="color: #663366;"><span style="background-image: linear-gradient(transparent, transparent), url(data:image/svg+xml; background-position: 100% 50%, 100% 50%; padding-right: 13px;">Primer3Plus</span></span> and <span style="color: #663366;"><span style="background-image: linear-gradient(transparent, transparent), url(data:image/svg+xml; background-position: 100% 50%, 100% 50%; padding-right: 13px;">PrimerQuest</span></span> can be used to find primers matching a wide variety of specifications. Highly degenerate primers for targeting a wide variety of DNA templates can be interactively designed using <span style="color: #663366;"><span style="background-image: linear-gradient(transparent, transparent), url(data:image/svg+xml; background-position: 100% 50%, 100% 50%; padding-right: 13px;">GeneFISHER</span></span>. Primers with high specificity for a subset of DNA templates in the presence of many similar variants can be designed using <span style="color: #663366;"><span style="background-image: linear-gradient(transparent, transparent), url(data:image/svg+xml; background-position: 100% 50%, 100% 50%; padding-right: 13px;">DECIPHER</span></span>.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> Mononucleotide and dinucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture (either other copies of same or the reverse direction primer); this phenomenon can lead to the production of 'primer dimer' products contaminating the mixture. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> When designing a primer for use in <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">TA cloning</span></span>, efficiency can be increased by adding AG tails to the 5' and the 3' end.<sup class="reference" id="cite_ref-6" style="line-height: 1; unicode-bidi: -webkit-isolate;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; white-space: nowrap;">[</span></span>6]</sup></div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> The reverse primer has to be the reverse complement of the given cDNA sequence. The reverse complement can be easily determined, e.g. with online calculators.<sup class="reference" id="cite_ref-7" style="line-height: 1; unicode-bidi: -webkit-isolate;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; white-space: nowrap;">[</span></span>7]</sup></div> <h3 style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-style: none; color: black; line-height: 1.6; margin: 0.3em 0px 0px; overflow: hidden; padding-bottom: 0px; padding-top: 0.5em;"> <span class="mw-headline" id="Degenerate_primers">Degenerate primers</span><span class="mw-editsection" style="-webkit-user-select: none; display: inline-block; font-size: x-small; font-weight: normal; line-height: 1em; margin-left: 1em; unicode-bidi: -webkit-isolate; vertical-align: baseline; white-space: nowrap;"><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">[</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">edit</span></span><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">]</span></span></h3> <div class="hatnote relarticle mainarticle" style="font-style: italic; margin-bottom: 0.5em; padding-left: 1.6em;"> Main article: <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Degenerate bases</span></span></div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> Sometimes <i>degenerate primers</i> are used. These are actually mixtures of similar, but not identical primers. They may be convenient if the same <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">gene</span></span> is to be amplified from different <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">organisms</span></span>, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">protein sequence</span></span>. As several different <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">codons</span></span> can code for one <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">amino acid</span></span>, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the<span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">amino acid</span></span> <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">isoleucine</span></span> might be "ATH", where A stands for <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">adenine</span></span>, T for <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">thymine</span></span>, and H for <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">adenine</span></span>, <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">thymine</span></span>, or <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">cytosine</span></span>, according to the <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">genetic code</span></span> for each <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">codon</span></span>, using the IUPAC symbols for <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">degenerate bases</span></span>. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using<span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">touchdown PCR</span></span>.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> <i>Degenerate primers</i> are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in <span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">GenBank</span></span>. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations.</div> <div style="line-height: inherit; margin-bottom: 0.5em; margin-top: 0.5em;"> There are a number of programs available to perform these primer predictions;</div> <ul style="line-height: 1.6; list-style-image: url(data:image/png; margin: 0.3em 0px 0px 1.6em; padding: 0px;"> <li style="margin-bottom: 0.1em;">CODEHOP can be run on a server though the server is no longer supported. It uses the block format.</li> <li style="margin-bottom: 0.1em;">HYDEN is an executable that runs on windows through command prompt.</li> <li style="margin-bottom: 0.1em;">iCODEHOP also uses the block format but has been taken down.</li> <li style="margin-bottom: 0.1em;">FAS-DPD (the most recent) has to be run through a Java operating system (JNODE) that can be set up in a virtual machine(VM).</li> </ul> <h2 style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 1px; color: black; font-family: 'Linux Libertine', Georgia, Times, serif; font-weight: normal; line-height: 1.3; margin: 1em 0px 0.25em; overflow: hidden; padding: 0px;"> <span class="mw-headline" id="See_also">See also</span><span class="mw-editsection" style="-webkit-user-select: none; display: inline-block; font-family: sans-serif; font-size: x-small; line-height: 1em; margin-left: 1em; unicode-bidi: -webkit-isolate; vertical-align: baseline; white-space: nowrap;"><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">[</span><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">edit</span></span><span class="mw-editsection-bracket" style="color: #555555; margin-left: 0px; margin-right: 0px;">]</span></span></h2> <ul style="line-height: 1.6; list-style-image: url(data:image/png; margin: 0.3em 0px 0px 1.6em; padding: 0px;"> <li style="margin-bottom: 0.1em;"><span style="color: #0b0080;"><span style="background-attachment: initial; background-clip: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Oligonucleotide synthesis</span></span> — the methods by which primers are manufactured</li> </ul> <h2 style="background-attachment: initial; background-clip: initial; background-image: none; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 1px; color: black; font-family: 'Linux Libertine', Georgia, Times, serif; font-weight: normal; line-height: 1.3; margin: 1em 0px 0.25em; overflow: hidden; padding: 0px;"> Source Courtesy: Wikipedia</h2> </div> </div> </div>

What is Primer ?

Primer (molecular biology) From Wikipedia, the free encyclopedia The DNA replication fork. RNA primer labeled at top. A ...

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What is PCR ? Polymerase chain reaction

<div dir="ltr" style="text-align: left;" trbidi="on"> <div class="noprint" id="mw-page-base"> </div> <div class="noprint" id="mw-head-base"> </div> <br /> <div class="mw-body" id="content" role="main"> <a href="https://www.blogger.com/null" id="top"></a> <div id="siteNotice"> <div id="centralNotice"> </div> </div> <h1 class="firstHeading" id="firstHeading" lang="en"> <span dir="auto">Polymerase chain reaction</span></h1> <div class="mw-body-content" id="bodyContent"> <div id="siteSub"> <br /></div> <div id="contentSub"> </div> <div class="mw-content-ltr" dir="ltr" id="mw-content-text" lang="en"> <div class="hatnote"> "PCR" redirects here. For other uses, see <a href="http://en.wikipedia.org/wiki/PCR_%28disambiguation%29" title="PCR (disambiguation)">PCR (disambiguation)</a>.</div> <table class="metadata plainlinks ambox ambox-move" role="presentation"> <tbody> <tr> <td class="mbox-image"> <div style="width: 52px;"> <img alt="" data-file-height="20" data-file-width="50" height="20" src="http://upload.wikimedia.org/wikipedia/commons/thumb/0/0f/Mergefrom.svg/50px-Mergefrom.svg.png" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/0/0f/Mergefrom.svg/75px-Mergefrom.svg.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/0/0f/Mergefrom.svg/100px-Mergefrom.svg.png 2x" width="50" /></div> </td> <td class="mbox-text"><span class="mbox-text-span">It has been suggested that <i><a href="http://en.wikipedia.org/wiki/Applications_of_PCR" title="Applications of PCR">Applications of PCR</a></i> be <a href="http://en.wikipedia.org/wiki/Wikipedia:Merging" title="Wikipedia:Merging">merged</a> into this article. (<a href="http://en.wikipedia.org/wiki/Talk:Polymerase_chain_reaction#Proposed_merge_with_Applications_of_PCR" title="Talk:Polymerase chain reaction">Discuss</a>) <small><i>Proposed since June 2013.</i></small></span></td> </tr> </tbody></table> <div class="thumb tright"> <div class="thumbinner" style="width: 302px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:PCR_tubes.png"><img alt="" class="thumbimage" data-file-height="656" data-file-width="800" height="246" src="http://upload.wikimedia.org/wikipedia/commons/thumb/8/81/PCR_tubes.png/300px-PCR_tubes.png" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/8/81/PCR_tubes.png/450px-PCR_tubes.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/8/81/PCR_tubes.png/600px-PCR_tubes.png 2x" width="300" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:PCR_tubes.png" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> A strip of eight PCR tubes, each containing a 100 μl reaction mixture</div> </div> </div> The <b>polymerase chain reaction</b> (<b>PCR</b>) is a biochemical technology in <a href="http://en.wikipedia.org/wiki/Molecular_biology" title="Molecular biology">molecular biology</a> used to <a href="http://en.wikipedia.org/wiki/DNA_replication" title="DNA replication">amplify</a> a single or a few copies of a piece of <a href="http://en.wikipedia.org/wiki/DNA" title="DNA">DNA</a> across several orders of magnitude, generating thousands to millions of copies of a particular <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_sequence" title="DNA sequence">DNA sequence</a>.<br /> Developed in 1983 by <a href="http://en.wikipedia.org/wiki/Kary_Mullis" title="Kary Mullis">Kary Mullis</a>,<sup class="reference" id="cite_ref-Bartlett_.26_Stirling_1-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Bartlett_.26_Stirling-1">[1]</a></sup><sup class="reference" id="cite_ref-pat_mar_2-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-pat_mar-2">[2]</a></sup> PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.<sup class="reference" id="cite_ref-Saiki1_3-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Saiki1-3">[3]</a></sup><sup class="reference" id="cite_ref-Saiki2_4-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Saiki2-4">[4]</a></sup> These include <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_cloning" title="DNA cloning">DNA cloning</a> for <a href="http://en.wikipedia.org/wiki/DNA_sequencing" title="DNA sequencing">sequencing</a>, DNA-based <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Phylogeny" title="Phylogeny">phylogeny</a>, or functional analysis of <a href="http://en.wikipedia.org/wiki/Gene" title="Gene">genes</a>; the diagnosis of <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Hereditary_disease" title="Hereditary disease">hereditary diseases</a>; the identification of <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Genetic_fingerprint" title="Genetic fingerprint">genetic fingerprints</a> (used in <a href="http://en.wikipedia.org/wiki/Forensic_science" title="Forensic science">forensic sciences</a> and <a href="http://en.wikipedia.org/wiki/Parental_testing" title="Parental testing">paternity testing</a>); and the detection and diagnosis of <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Infectious_disease" title="Infectious disease">infectious diseases</a>. In 1993, Mullis was awarded the <a href="http://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistry" title="Nobel Prize in Chemistry">Nobel Prize in Chemistry</a> along with <a href="http://en.wikipedia.org/wiki/Michael_Smith_%28chemist%29" title="Michael Smith (chemist)">Michael Smith</a> for his work on PCR.<sup class="reference" id="cite_ref-Kary_Mullis_Nobel_Lecture_5-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Kary_Mullis_Nobel_Lecture-5">[5]</a></sup><br /> The method relies on <a href="http://en.wikipedia.org/wiki/Thermal_cycler" title="Thermal cycler">thermal cycling</a>, consisting of cycles of repeated heating and cooling of the reaction for <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_melting" title="DNA melting">DNA melting</a> and <a href="http://en.wikipedia.org/wiki/Enzyme" title="Enzyme">enzymatic</a> <a href="http://en.wikipedia.org/wiki/DNA_replication" title="DNA replication">replication</a> of the DNA. <a href="http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29" title="Primer (molecular biology)">Primers</a> (short DNA fragments) containing sequences complementary to the target region along with a <a href="http://en.wikipedia.org/wiki/DNA_polymerase" title="DNA polymerase">DNA polymerase</a> (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a <a href="http://en.wikipedia.org/wiki/Chain_reaction" title="Chain reaction">chain reaction</a> in which the DNA template is <a href="http://en.wikipedia.org/wiki/Exponential_growth" title="Exponential growth">exponentially</a> amplified. PCR can be extensively modified to perform a wide array of <a href="http://en.wikipedia.org/wiki/Genetic_engineering" title="Genetic engineering">genetic manipulations</a>.<br /> Almost all PCR applications employ a heat-stable DNA polymerase, such as <a href="http://en.wikipedia.org/wiki/Taq_polymerase" title="Taq polymerase">Taq polymerase</a> (an enzyme originally isolated from the bacterium <i><a href="http://en.wikipedia.org/wiki/Thermus_aquaticus" title="Thermus aquaticus">Thermus aquaticus</a></i>). This DNA polymerase <a href="http://en.wikipedia.org/wiki/Enzyme" title="Enzyme">enzymatically</a> assembles a new DNA strand from DNA building-blocks, the <a href="http://en.wikipedia.org/wiki/Nucleotide" title="Nucleotide">nucleotides</a>, by using single-stranded DNA as a template and DNA <a href="http://en.wikipedia.org/wiki/Oligonucleotide" title="Oligonucleotide">oligonucleotides</a> (also called <a href="http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29" title="Primer (molecular biology)">DNA primers</a>), which are required for initiation of DNA synthesis. The vast majority of PCR methods use <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Thermocycler" title="Thermocycler">thermal cycling</a>, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps.<br /> In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_melting" title="DNA melting">DNA melting</a>. In the second step, the temperature is lowered and the two DNA strands become <a href="http://en.wikipedia.org/wiki/DNA#Polymerases" title="DNA">templates</a> for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of <a href="http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29" title="Primer (molecular biology)">primers</a> that are <a href="http://en.wikipedia.org/wiki/Complementary_DNA" title="Complementary DNA">complementary</a> to the DNA region targeted for amplification under specific thermal cycling conditions.<br /> <div class="thumb tright"> <div class="thumbinner" style="width: 302px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:PCR_masina_kasutamine.jpg"><img alt="" class="thumbimage" data-file-height="2000" data-file-width="3008" height="199" src="http://upload.wikimedia.org/wikipedia/commons/thumb/6/67/PCR_masina_kasutamine.jpg/300px-PCR_masina_kasutamine.jpg" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/6/67/PCR_masina_kasutamine.jpg/450px-PCR_masina_kasutamine.jpg 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/6/67/PCR_masina_kasutamine.jpg/600px-PCR_masina_kasutamine.jpg 2x" width="300" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:PCR_masina_kasutamine.jpg" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> Placing a strip of eight PCR tubes, each containing a 100 μl reaction mixture, into the thermal cycler</div> </div> </div> <br /> <div class="toc" id="toc"> <div id="toctitle"> <h2> Contents</h2> </div> <ul> <li class="toclevel-1 tocsection-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#PCR_principles_and_procedure"><span class="tocnumber">1</span> <span class="toctext">PCR principles and procedure</span></a> <ul> <li class="toclevel-2 tocsection-2"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Procedure"><span class="tocnumber">1.1</span> <span class="toctext">Procedure</span></a></li> </ul> </li> <li class="toclevel-1 tocsection-3"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#PCR_stages"><span class="tocnumber">2</span> <span class="toctext">PCR stages</span></a> <ul> <li class="toclevel-2 tocsection-4"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#PCR_optimization"><span class="tocnumber">2.1</span> <span class="toctext">PCR optimization</span></a></li> </ul> </li> <li class="toclevel-1 tocsection-5"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Application_of_PCR"><span class="tocnumber">3</span> <span class="toctext">Application of PCR</span></a> <ul> <li class="toclevel-2 tocsection-6"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Selective_DNA_isolation"><span class="tocnumber">3.1</span> <span class="toctext">Selective DNA isolation</span></a></li> <li class="toclevel-2 tocsection-7"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Amplification_and_quantification_of_DNA"><span class="tocnumber">3.2</span> <span class="toctext">Amplification and quantification of DNA</span></a></li> <li class="toclevel-2 tocsection-8"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#PCR_in_diagnosis_of_diseases"><span class="tocnumber">3.3</span> <span class="toctext">PCR in diagnosis of diseases</span></a></li> </ul> </li> <li class="toclevel-1 tocsection-9"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Variations_on_the_basic_PCR_technique"><span class="tocnumber">4</span> <span class="toctext">Variations on the basic PCR technique</span></a></li> <li class="toclevel-1 tocsection-10"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#History"><span class="tocnumber">5</span> <span class="toctext">History</span></a> <ul> <li class="toclevel-2 tocsection-11"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Patent_disputes"><span class="tocnumber">5.1</span> <span class="toctext">Patent disputes</span></a></li> </ul> </li> <li class="toclevel-1 tocsection-12"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#References"><span class="tocnumber">6</span> <span class="toctext">References</span></a></li> <li class="toclevel-1 tocsection-13"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#External_links"><span class="tocnumber">7</span> <span class="toctext">External links</span></a></li> </ul> </div> <br /> <h2> <span class="mw-headline" id="PCR_principles_and_procedure">PCR principles and procedure</span></h2> <div class="thumb tright"> <div class="thumbinner" style="width: 202px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:G-Storm_thermal_cycler.jpg"><img alt="" class="thumbimage" data-file-height="1200" data-file-width="1600" height="150" src="http://upload.wikimedia.org/wikipedia/commons/thumb/b/b8/G-Storm_thermal_cycler.jpg/200px-G-Storm_thermal_cycler.jpg" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/b/b8/G-Storm_thermal_cycler.jpg/300px-G-Storm_thermal_cycler.jpg 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/b/b8/G-Storm_thermal_cycler.jpg/400px-G-Storm_thermal_cycler.jpg 2x" width="200" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:G-Storm_thermal_cycler.jpg" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> <b>Figure 1a</b>: A thermal cycler for PCR</div> </div> </div> <div class="thumb tright"> <div class="thumbinner" style="width: 222px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:Primitive_PCR_machine_for_scrap.JPG"><img alt="" class="thumbimage" data-file-height="1264" data-file-width="1122" height="248" src="http://upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Primitive_PCR_machine_for_scrap.JPG/220px-Primitive_PCR_machine_for_scrap.JPG" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Primitive_PCR_machine_for_scrap.JPG/330px-Primitive_PCR_machine_for_scrap.JPG 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Primitive_PCR_machine_for_scrap.JPG/440px-Primitive_PCR_machine_for_scrap.JPG 2x" width="220" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:Primitive_PCR_machine_for_scrap.JPG" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> <b>Figure 1b</b>: An older model three-temperature thermal cycler for PCR</div> </div> </div> PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of between 0.1 and 10 <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Kilo_base_pair" title="Kilo base pair">kilo base pairs</a> (kb), although some techniques allow for amplification of fragments up to 40 kb in size.<sup class="reference" id="cite_ref-6"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-6">[6]</a></sup> The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses.<sup class="reference" id="cite_ref-7"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-7">[7]</a></sup><br /> A basic PCR set up requires several components and reagents.<sup class="reference" id="cite_ref-molecular_cloning_8-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-molecular_cloning-8">[8]</a></sup> These components include:<br /> <ul> <li><i>DNA template</i> that contains the DNA region (target) to be amplified.</li> <li>Two <i><a href="http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29" title="Primer (molecular biology)">primers</a></i> that are <a href="http://en.wikipedia.org/wiki/Complementarity_%28molecular_biology%29" title="Complementarity (molecular biology)">complementary</a> to the <a href="http://en.wikipedia.org/wiki/Directionality_%28molecular_biology%29" title="Directionality (molecular biology)">3'</a> (three prime) ends of each of the <a href="http://en.wikipedia.org/wiki/DNA#Sense_and_antisense" title="DNA">sense and anti-sense</a> strand of the DNA target.</li> <li><i><a href="http://en.wikipedia.org/wiki/Taq_polymerase" title="Taq polymerase">Taq polymerase</a></i> or another <a href="http://en.wikipedia.org/wiki/DNA_polymerase" title="DNA polymerase">DNA polymerase</a> with a temperature optimum at around 70 °C.</li> <li><i>Deoxynucleoside triphosphates</i> (dNTPs, sometimes called "deoxynucleotide triphosphates"; <a href="http://en.wikipedia.org/wiki/Nucleotide" title="Nucleotide">nucleotides</a> containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.</li> <li><i><a href="http://en.wikipedia.org/wiki/Buffer_solution" title="Buffer solution">Buffer solution</a></i>, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.</li> <li><i><a href="http://en.wikipedia.org/wiki/Bivalent" title="Bivalent">Bivalent</a> <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Cations" title="Cations">cations</a></i>, <a href="http://en.wikipedia.org/wiki/Magnesium" title="Magnesium">magnesium</a> or <a href="http://en.wikipedia.org/wiki/Manganese" title="Manganese">manganese</a> ions; generally Mg²⁺ is used, but Mn²⁺ can be utilized for PCR-mediated DNA <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PCR_mutagenesis" title="PCR mutagenesis">mutagenesis</a>, as higher Mn²⁺ concentration increases the error rate during DNA synthesis<sup class="reference" id="cite_ref-9"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-9">[9]</a></sup></li> <li><i>Monovalent cation</i> <a href="http://en.wikipedia.org/wiki/Potassium" title="Potassium">potassium</a> ions.</li> </ul> The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a <a href="http://en.wikipedia.org/wiki/Thermal_cycler" title="Thermal cycler">thermal cycler</a>. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the <a href="http://en.wikipedia.org/wiki/Thermoelectric_cooling" title="Thermoelectric cooling">Peltier effect</a>, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.<br /> <h3> <span class="mw-headline" id="Procedure">Procedure</span></h3> Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Figure below). The cycling is often preceded by a single temperature step (called <i>hold</i>) at a high temperature (>90 °C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (<a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_melting" title="DNA melting">Tm</a>) of the primers.<sup class="reference" id="cite_ref-10"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-10">[10]</a></sup><br /> <ul> <li><i>Initialization step</i>: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by <a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Variations_on_the_basic_PCR_technique" title="Polymerase chain reaction">hot-start PCR</a>.<sup class="reference" id="cite_ref-antibody_hot_start_11-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-antibody_hot_start-11">[11]</a></sup></li> <li><i><a href="http://en.wikipedia.org/wiki/Denaturation_%28biochemistry%29#Nucleic_acid_denaturation" title="Denaturation (biochemistry)">Denaturation step</a></i>: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_melting" title="DNA melting">DNA melting</a> of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.</li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Annealing_%28biology%29" title="Annealing (biology)">Annealing step</a></i>: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3–5 °C below the Tm of the primers used. Stable DNA–DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation.</li> <li><i>Extension/elongation step</i>: The temperature at this step depends on the DNA polymerase used; <a href="http://en.wikipedia.org/wiki/Taq_polymerase" title="Taq polymerase">Taq polymerase</a> has its optimum <a href="http://en.wikipedia.org/wiki/Enzyme" title="Enzyme">activity</a> temperature at 75–80 °C,<sup class="reference" id="cite_ref-Chien_et_al._12-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Chien_et_al.-12">[12]</a></sup><sup class="reference" id="cite_ref-Lawyer_et_al._13-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Lawyer_et_al.-13">[13]</a></sup> and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-<a class="mw-redirect" href="http://en.wikipedia.org/wiki/Phosphate_group" title="Phosphate group">phosphate group</a> of the dNTPs with the 3'-<a class="mw-redirect" href="http://en.wikipedia.org/wiki/Hydroxyl_group" title="Hydroxyl group">hydroxyl group</a> at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.</li> <li><i>Final elongation</i>: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.</li> <li><i>Final hold</i>: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.</li> </ul> <a class="image" href="http://en.wikipedia.org/wiki/File:Polymerase_chain_reaction.svg" title="Figure 2: Schematic drawing of the PCR cycle."><img alt="Figure 2: Schematic drawing of the PCR cycle." data-file-height="439" data-file-width="842" height="438" src="http://upload.wikimedia.org/wikipedia/commons/thumb/9/96/Polymerase_chain_reaction.svg/840px-Polymerase_chain_reaction.svg.png" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/9/96/Polymerase_chain_reaction.svg/1260px-Polymerase_chain_reaction.svg.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/9/96/Polymerase_chain_reaction.svg/1680px-Polymerase_chain_reaction.svg.png 2x" width="840" /></a><br /> <div class="thumb tright"> <div class="thumbinner" style="width: 302px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:Roland_Gel.JPG"><img alt="" class="thumbimage" data-file-height="488" data-file-width="518" height="283" src="http://upload.wikimedia.org/wikipedia/commons/thumb/d/d0/Roland_Gel.JPG/300px-Roland_Gel.JPG" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/d/d0/Roland_Gel.JPG/450px-Roland_Gel.JPG 1.5x, //upload.wikimedia.org/wikipedia/commons/d/d0/Roland_Gel.JPG 2x" width="300" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:Roland_Gel.JPG" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> <b>Figure 3</b>: Ethidium bromide-stained PCR products after <a href="http://en.wikipedia.org/wiki/Gel_electrophoresis" title="Gel electrophoresis">gel electrophoresis</a>. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.</div> </div> </div> To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or <a href="http://en.wikipedia.org/wiki/Amplicon" title="Amplicon">amplicon</a>), <a href="http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis" title="Agarose gel electrophoresis">agarose gel electrophoresis</a> is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_ladder" title="DNA ladder">DNA ladder</a> (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).<br /> <h2> <span class="mw-headline" id="PCR_stages">PCR stages</span></h2> The PCR process can be divided into three stages:<br /> <i>Exponential amplification</i>: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.<sup class="reference" id="cite_ref-14"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-14">[14]</a></sup><br /> <i>Leveling off stage</i>: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.<br /> <i>Plateau</i>: No more product accumulates due to exhaustion of reagents and enzyme.<br /> <h3> <span class="mw-headline" id="PCR_optimization">PCR optimization</span></h3> <div class="hatnote relarticle mainarticle"> Main article: <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PCR_optimization" title="PCR optimization">PCR optimization</a></div> In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.<sup class="reference" id="cite_ref-15"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-15">[15]</a></sup><sup class="reference" id="cite_ref-16"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-16">[16]</a></sup> Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.<sup class="reference" id="cite_ref-molecular_cloning_8-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-molecular_cloning-8">[8]</a></sup> This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as <a href="http://en.wikipedia.org/wiki/Formamide" title="Formamide">formamide</a>, in buffer systems may increase the specificity and yield of PCR.<sup class="reference" id="cite_ref-17"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-17">[17]</a></sup> Computer simulations of theoretical PCR results (<a href="http://en.wikipedia.org/wiki/In_silico_PCR" title="In silico PCR">Electronic PCR</a>) may be performed to assist in primer design.<sup class="reference" id="cite_ref-ePCR_18-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-ePCR-18">[18]</a></sup><br /> <h2> <span class="mw-headline" id="Application_of_PCR">Application of PCR</span></h2> <div class="hatnote relarticle mainarticle"> Main article: <a href="http://en.wikipedia.org/wiki/Applications_of_PCR" title="Applications of PCR">Applications of PCR</a></div> <h3> <span class="mw-headline" id="Selective_DNA_isolation">Selective DNA isolation</span></h3> PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating <a href="http://en.wikipedia.org/wiki/Hybridization_probe" title="Hybridization probe">hybridization probes</a> for <a href="http://en.wikipedia.org/wiki/Southern_blot" title="Southern blot">Southern</a> or <a href="http://en.wikipedia.org/wiki/Northern_blot" title="Northern blot">northern</a> hybridization and <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_cloning" title="DNA cloning">DNA cloning</a>, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.<br /> Other applications of PCR include <a href="http://en.wikipedia.org/wiki/DNA_sequencing" title="DNA sequencing">DNA sequencing</a> to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite <a href="http://en.wikipedia.org/wiki/Recombinant_DNA" title="Recombinant DNA">recombinant DNA</a> technologies involving the insertion of a DNA sequence into a <a href="http://en.wikipedia.org/wiki/Plasmid" title="Plasmid">plasmid</a> or the genetic material of another organism. Bacterial colonies (<a href="http://en.wikipedia.org/wiki/Escherichia_coli" title="Escherichia coli">E. coli</a>) can be rapidly screened by PCR for correct DNA <a href="http://en.wikipedia.org/wiki/Plasmid" title="Plasmid">vector</a> constructs.<sup class="reference" id="cite_ref-Hybrid_19-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Hybrid-19">[19]</a></sup> PCR may also be used for <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Genetic_fingerprinting" title="Genetic fingerprinting">genetic fingerprinting</a>; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.<br /> Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms.<sup class="noprint Inline-Template Template-Fact" style="white-space: nowrap;">[<i><a href="http://en.wikipedia.org/wiki/Wikipedia:Citation_needed" title="Wikipedia:Citation needed"><span title="This claim needs references to reliable sources. (January 2011)">citation needed</span></a></i>]</sup><br /> <div class="thumb tright"> <div class="thumbinner" style="width: 202px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:Pcr_fingerprint.png"><img alt="" class="thumbimage" data-file-height="498" data-file-width="331" height="301" src="http://upload.wikimedia.org/wikipedia/commons/thumb/0/02/Pcr_fingerprint.png/200px-Pcr_fingerprint.png" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/0/02/Pcr_fingerprint.png/300px-Pcr_fingerprint.png 1.5x, //upload.wikimedia.org/wikipedia/commons/0/02/Pcr_fingerprint.png 2x" width="200" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:Pcr_fingerprint.png" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> <b>Figure 4</b>: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.</div> </div> </div> <h3> <span class="mw-headline" id="Amplification_and_quantification_of_DNA">Amplification and quantification of DNA</span></h3> Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Forensic_analysis" title="Forensic analysis">forensic analysis</a>, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of <a href="http://en.wikipedia.org/wiki/Ancient_DNA" title="Ancient DNA">ADNA</a> that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old <a href="http://en.wikipedia.org/wiki/Mammoth" title="Mammoth">mammoth</a>, and also on human DNA, in applications ranging from the analysis of Egyptian <a href="http://en.wikipedia.org/wiki/Mummy" title="Mummy">mummies</a> to the identification of a <a href="http://en.wikipedia.org/wiki/Russia" title="Russia">Russian</a> <a href="http://en.wikipedia.org/wiki/Tsar" title="Tsar">tsar</a> and the body of English king <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Richard_III" title="Richard III">Richard III</a>.<sup class="reference" id="cite_ref-20"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-20">[20]</a></sup><br /> Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of <a href="http://en.wikipedia.org/wiki/Gene_expression" title="Gene expression">gene expression</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Quantitative_PCR" title="Quantitative PCR">Quantitative PCR</a> is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.<br /> <div class="hatnote boilerplate seealso"> See also: <a href="http://en.wikipedia.org/wiki/Use_of_DNA_in_forensic_entomology" title="Use of DNA in forensic entomology">Use of DNA in forensic entomology</a></div> <h3> <span class="mw-headline" id="PCR_in_diagnosis_of_diseases">PCR in diagnosis of diseases</span></h3> PCR permits early diagnosis of <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Malignant" title="Malignant">malignant</a> diseases such as <a href="http://en.wikipedia.org/wiki/Leukemia" title="Leukemia">leukemia</a> and <a href="http://en.wikipedia.org/wiki/Lymphoma" title="Lymphoma">lymphomas</a>, which is currently the highest-developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods.<sup class="noprint Inline-Template Template-Fact" style="white-space: nowrap;">[<i><a href="http://en.wikipedia.org/wiki/Wikipedia:Citation_needed" title="Wikipedia:Citation needed"><span title="This claim needs references to reliable sources. (May 2008)">citation needed</span></a></i>]</sup><br /> PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.<sup class="reference" id="cite_ref-Cai2014_21-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Cai2014-21">[21]</a></sup> PCR also permits identification of non-cultivatable or slow-growing microorganisms such as <a href="http://en.wikipedia.org/wiki/Mycobacterium" title="Mycobacterium">mycobacteria</a>, <a href="http://en.wikipedia.org/wiki/Anaerobic_organism" title="Anaerobic organism">anaerobic bacteria</a>, or <a href="http://en.wikipedia.org/wiki/Virus" title="Virus">viruses</a> from <a href="http://en.wikipedia.org/wiki/Tissue_culture" title="Tissue culture">tissue culture</a> assays and <a href="http://en.wikipedia.org/wiki/Animal_model" title="Animal model">animal models</a>. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.<sup class="reference" id="cite_ref-Cai2014_21-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Cai2014-21">[21]</a></sup><sup class="reference" id="cite_ref-22"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-22">[22]</a></sup><br /> Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease.<sup class="reference" id="cite_ref-Cai2014_21-2"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Cai2014-21">[21]</a></sup> Such early detection may give physicians a significant lead time in treatment. The amount of virus ("<a href="http://en.wikipedia.org/wiki/Viral_load" title="Viral load">viral load</a>") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).<br /> <h2> <span class="mw-headline" id="Variations_on_the_basic_PCR_technique">Variations on the basic PCR technique</span></h2> <div class="hatnote relarticle mainarticle"> Main article: <a href="http://en.wikipedia.org/wiki/Variants_of_PCR" title="Variants of PCR">Variants of PCR</a></div> <ul> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Allele-specific_PCR&action=edit&redlink=1" title="Allele-specific PCR (page does not exist)">Allele-specific PCR</a></i>: a diagnostic or cloning technique based on single-nucleotide variations (SNVs not to be confused with <a href="http://en.wikipedia.org/wiki/Single-nucleotide_polymorphism" title="Single-nucleotide polymorphism">SNPs</a>) (single-base differences in a patient). It requires prior knowledge of a DNA sequence, including differences between <a href="http://en.wikipedia.org/wiki/Allele" title="Allele">alleles</a>, and uses primers whose 3' ends encompass the SNV (base pair buffer around SNV usually incorporated). PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.<sup class="reference" id="cite_ref-23"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-23">[23]</a></sup> See <a href="http://en.wikipedia.org/wiki/SNP_genotyping" title="SNP genotyping">SNP genotyping</a> for more information.</li> <li><i><a href="http://en.wikipedia.org/wiki/Polymerase_cycling_assembly" title="Polymerase cycling assembly">Assembly PCR</a></i> or <i>Polymerase Cycling Assembly (PCA)</i>: artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.<sup class="reference" id="cite_ref-Stemmer_et_al._24-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Stemmer_et_al.-24">[24]</a></sup></li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Asymmetric_PCR&action=edit&redlink=1" title="Asymmetric PCR (page does not exist)">Asymmetric PCR</a></i>: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in <a href="http://en.wikipedia.org/wiki/Sequencing" title="Sequencing">sequencing</a> and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (<a href="http://en.wikipedia.org/wiki/Arithmetic" title="Arithmetic">arithmetic</a>) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.<sup class="reference" id="cite_ref-Innis_et_al._25-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Innis_et_al.-25">[25]</a></sup> A recent modification on this process, known as <i>L</i>inear-<i>A</i>fter-<i>T</i>he-<i>E</i>xponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (<a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_melting" title="DNA melting">Tm</a>) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.<sup class="reference" id="cite_ref-Pierce_and_Wangh_26-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Pierce_and_Wangh-26">[26]</a></sup></li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Dial-out_PCR&action=edit&redlink=1" title="Dial-out PCR (page does not exist)">Dial-out PCR</a></i>: a highly parallel method for retrieving accurate DNA molecules for gene synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR.<sup class="reference" id="cite_ref-Schwartz_et_al._27-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Schwartz_et_al.-27">[27]</a></sup></li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Digital_PCR" title="Digital PCR">Digital PCR</a> (dPCR)</i>: used to measure the quantity of a target DNA sequence in a DNA sample. The DNA sample is highly diluted so that after running many PCRs in parallel, some of them will not receive a single molecule of the target DNA. The target DNA concentration is calculated using the proportion of negative outcomes. Hence the name 'digital PCR'.</li> <li><i><a href="http://en.wikipedia.org/wiki/Helicase-dependent_amplification" title="Helicase-dependent amplification">Helicase-dependent amplification</a></i>: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_helicase" title="DNA helicase">DNA helicase</a>, an enzyme that unwinds DNA, is used in place of thermal denaturation.<sup class="reference" id="cite_ref-28"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-28">[28]</a></sup></li> <li><i><a href="http://en.wikipedia.org/wiki/Hot_start_PCR" title="Hot start PCR">Hot start PCR</a></i>: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95 °C) before adding the polymerase.<sup class="reference" id="cite_ref-general_hot_start_29-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-general_hot_start-29">[29]</a></sup> Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an <a href="http://en.wikipedia.org/wiki/Antibody" title="Antibody">antibody</a><sup class="reference" id="cite_ref-antibody_hot_start_11-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-antibody_hot_start-11">[11]</a></sup><sup class="reference" id="cite_ref-antibody_hot_start_2_30-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-antibody_hot_start_2-30">[30]</a></sup> or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.</li> <li><i><a href="http://en.wikipedia.org/wiki/In_silico_PCR" title="In silico PCR">In silico PCR</a></i> (digital PCR, virtual PCR, electronic PCR, e-PCR) refers to computational tools used to calculate theoretical polymerase chain reaction results using a given set of <a href="http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29" title="Primer (molecular biology)">primers</a> (<a href="http://en.wikipedia.org/wiki/Hybridization_probe" title="Hybridization probe">probes</a>) to amplify <a href="http://en.wikipedia.org/wiki/DNA" title="DNA">DNA</a> sequences from a sequenced <a href="http://en.wikipedia.org/wiki/Genome" title="Genome">genome</a> or <a href="http://en.wikipedia.org/wiki/Transcriptome" title="Transcriptome">transcriptome</a>. In silico PCR was proposed as an educational tool for molecular biology.<sup class="reference" id="cite_ref-31"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-31">[31]</a></sup></li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Intersequence-specific_PCR&action=edit&redlink=1" title="Intersequence-specific PCR (page does not exist)">Intersequence-specific PCR</a></i> (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.<sup class="reference" id="cite_ref-32"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-32">[32]</a></sup></li> <li><i><a href="http://en.wikipedia.org/wiki/Inverse_polymerase_chain_reaction" title="Inverse polymerase chain reaction">Inverse PCR</a></i>: is commonly used to identify the flanking sequences around <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Genomic" title="Genomic">genomic</a> inserts. It involves a series of <a href="http://en.wikipedia.org/wiki/Restriction_digest" title="Restriction digest">DNA digestions</a> and <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Self_ligation" title="Self ligation">self ligation</a>, resulting in known sequences at either end of the unknown sequence.<sup class="reference" id="cite_ref-Ochman_et_al._33-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Ochman_et_al.-33">[33]</a></sup></li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Ligation-mediated_PCR&action=edit&redlink=1" title="Ligation-mediated PCR (page does not exist)">Ligation-mediated PCR</a></i>: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for <a href="http://en.wikipedia.org/wiki/DNA_sequencing" title="DNA sequencing">DNA sequencing</a>, <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Genome_walking" title="Genome walking">genome walking</a>, and <a href="http://en.wikipedia.org/wiki/DNA_footprinting" title="DNA footprinting">DNA footprinting</a>.<sup class="reference" id="cite_ref-Mueller_and_Wold_34-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Mueller_and_Wold-34">[34]</a></sup></li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Methylation-specific_PCR" title="Methylation-specific PCR">Methylation-specific PCR</a></i> (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,<sup class="reference" id="cite_ref-35"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-35">[35]</a></sup> and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.</li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Miniprimer_PCR&action=edit&redlink=1" title="Miniprimer PCR (page does not exist)">Miniprimer PCR</a></i>: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.<sup class="reference" id="cite_ref-Isenbarger_.27.27et_al..27.27._36-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Isenbarger_.27.27et_al..27.27.-36">[36]</a></sup></li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Multiplex_Ligation-dependent_Probe_Amplification" title="Multiplex Ligation-dependent Probe Amplification">Multiplex Ligation-dependent Probe Amplification</a></i> (<i>MLPA</i>): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).</li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Multiplex-PCR" title="Multiplex-PCR">Multiplex-PCR</a></i>: consists of multiple primer sets within a single PCR mixture to produce <a href="http://en.wikipedia.org/wiki/Amplicon" title="Amplicon">amplicons</a> of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by <a href="http://en.wikipedia.org/wiki/Gel_electrophoresis" title="Gel electrophoresis">gel electrophoresis</a>.</li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Nanoparticle-Assisted_PCR_%28nanoPCR%29&action=edit&redlink=1" title="Nanoparticle-Assisted PCR (nanoPCR) (page does not exist)">Nanoparticle-Assisted PCR (nanoPCR)</a></i>: In recent years, it has been reported that some nanoparticles (NPs) can enhance the efficiency of PCR (thus being called nanoPCR), and some even perform better than the original PCR enhancers. It was also found that quantum dots (QDs) can improve PCR specificity and efficiency. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are efficient in enhancing the amplification of long PCR. Carbon nanopowder (CNP) was reported be able to improve the efficiency of repeated PCR and long PCR. ZnO, TiO<small>2</small>, and Ag NPs were also found to increase PCR yield. Importantly, already known data has indicated that non-metallic NPs retained acceptable amplification fidelity. Given that many NPs are capable of enhancing PCR efficiency, it is clear that there is likely to be great potential for nanoPCR technology improvements and product development.<sup class="reference" id="cite_ref-37"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-37">[37]</a></sup><sup class="reference" id="cite_ref-38"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-38">[38]</a></sup></li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Nested_PCR" title="Nested PCR">Nested PCR</a></i>: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.</li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Overlap-extension_PCR" title="Overlap-extension PCR">Overlap-extension PCR</a></i> or <i>Splicing by overlap extension (SOEing)</i> : a <a href="http://en.wikipedia.org/wiki/Genetic_engineering" title="Genetic engineering">genetic engineering</a> technique that is used to splice together two or more DNA fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations; the technique enables creation of specific and long DNA constructs. It can also introduce deletions, insertions or point mutations into a DNA sequence.<sup class="reference" id="cite_ref-39"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-39">[39]</a></sup><sup class="reference" id="cite_ref-40"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-40">[40]</a></sup></li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=PAN-AC&action=edit&redlink=1" title="PAN-AC (page does not exist)">PAN-AC</a></i>: uses isothermal conditions for amplification, and may be used in living cells.<sup class="reference" id="cite_ref-41"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-41">[41]</a></sup><sup class="reference" id="cite_ref-42"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-42">[42]</a></sup></li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Quantitative_PCR" title="Quantitative PCR">quantitative PCR</a></i> (qPCR): used to measure the quantity of a target sequence (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Quantitative_PCR" title="Quantitative PCR">quantitative PCR</a> is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. <i>Quantitative PCR</i> has a very high degree of precision. Quantitative PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or <a href="http://en.wikipedia.org/wiki/Fluorophore" title="Fluorophore">fluorophore</a>-containing DNA probes, such as <a href="http://en.wikipedia.org/wiki/TaqMan" title="TaqMan">TaqMan</a>, to measure the amount of amplified product in real time. It is also sometimes abbreviated to <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Quantitative_PCR" title="Quantitative PCR">RT-PCR</a> (<i>real-time</i> PCR) but this abbreviation should be used only for <a href="http://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction" title="Reverse transcription polymerase chain reaction">reverse transcription PCR</a>. qPCR is the appropriate contractions for <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Quantitative_PCR" title="Quantitative PCR">quantitative PCR</a> (real-time PCR).</li> <li><i>Reverse Transcription PCR (<a class="mw-redirect" href="http://en.wikipedia.org/wiki/RT-PCR" title="RT-PCR">RT-PCR</a>)</i>: for amplifying DNA from RNA. <a href="http://en.wikipedia.org/wiki/Reverse_transcriptase" title="Reverse transcriptase">Reverse transcriptase</a> reverse transcribes <a href="http://en.wikipedia.org/wiki/RNA" title="RNA">RNA</a> into <a class="mw-redirect" href="http://en.wikipedia.org/wiki/CDNA" title="CDNA">cDNA</a>, which is then amplified by PCR. RT-PCR is widely used in <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Expression_profiling" title="Expression profiling">expression profiling</a>, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Exons" title="Exons">exons</a> and <a class="mw-redirect" href="http://en.wikipedia.org/wiki/Introns" title="Introns">introns</a> in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by <a class="mw-redirect" href="http://en.wikipedia.org/wiki/RACE_%28biology%29" title="RACE (biology)">RACE-PCR</a> (<i>Rapid Amplification of cDNA Ends</i>).</li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Solid_Phase_PCR&action=edit&redlink=1" title="Solid Phase PCR (page does not exist)">Solid Phase PCR</a></i>: encompasses multiple meanings, including <a href="http://en.wikipedia.org/wiki/Polony_%28biology%29" title="Polony (biology)">Polony Amplification</a> (where PCR colonies are derived in a gel matrix, for example), Bridge PCR<sup class="reference" id="cite_ref-43"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-43">[43]</a></sup> (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR<sup class="reference" id="cite_ref-44"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-44">[44]</a></sup> (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).</li> </ul> <ul> <li><i>Suicide PCR</i>: typically used in <a href="http://en.wikipedia.org/wiki/Paleogenetics" title="Paleogenetics">paleogenetics</a> or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is the highest priority. It was originally described in a study to verify the presence of the microbe <a href="http://en.wikipedia.org/wiki/Yersinia_pestis" title="Yersinia pestis">Yersinia pestis</a> in dental samples obtained from 14th Century graves of people supposedly killed by plague during the medieval <a href="http://en.wikipedia.org/wiki/Black_Death" title="Black Death">Black Death</a> epidemic.<sup class="reference" id="cite_ref-45"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-45">[45]</a></sup> The method prescribes the use of any primer combination only once in a PCR (hence the term "suicide"), which should never have been used in any positive control PCR reaction, and the primers should always target a genomic region never amplified before in the lab using this or any other set of primers. This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives.</li> </ul> <ul> <li><i>Thermal asymmetric interlaced PCR (<a class="new" href="http://en.wikipedia.org/w/index.php?title=TAIL-PCR&action=edit&redlink=1" title="TAIL-PCR (page does not exist)">TAIL-PCR</a>)</i>: for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.<sup class="reference" id="cite_ref-46"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-46">[46]</a></sup></li> <li><i><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Touchdown_PCR" title="Touchdown PCR">Touchdown PCR</a></i> (<i>Step-down PCR</i>): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5 °C) above the T_m of the primers used, while at the later cycles, it is a few degrees (3-5 °C) below the primer T_m. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.<sup class="reference" id="cite_ref-47"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-47">[47]</a></sup></li> <li><i><a class="new" href="http://en.wikipedia.org/w/index.php?title=Universal_Fast_Walking&action=edit&redlink=1" title="Universal Fast Walking (page does not exist)">Universal Fast Walking</a></i>: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer — which can lead to artefactual 'noise')<sup class="reference" id="cite_ref-Myrick_and_Gelbart_48-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Myrick_and_Gelbart-48">[48]</a></sup> by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),<sup class="reference" id="cite_ref-lane_rage_49-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-lane_rage-49">[49]</a></sup> 5'RACE LaNe<sup class="reference" id="cite_ref-Parkb_50-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Parkb-50">[50]</a></sup> and 3'RACE LaNe.<sup class="reference" id="cite_ref-Park_51-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Park-51">[51]</a></sup></li> </ul> <h2> <span class="mw-headline" id="History">History</span></h2> <div class="thumb tright"> <div class="thumbinner" style="width: 222px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:Primers_RevComp.svg"><img alt="" class="thumbimage" data-file-height="536" data-file-width="1028" height="115" src="http://upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_RevComp.svg/220px-Primers_RevComp.svg.png" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_RevComp.svg/330px-Primers_RevComp.svg.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_RevComp.svg/440px-Primers_RevComp.svg.png 2x" width="220" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:Primers_RevComp.svg" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> Diagrammatic representation of an example primer pair. The use of primers in an in vitro assay to allow DNA synthesis was a major innovation which allowed the development of PCR.</div> </div> </div> <div class="hatnote relarticle mainarticle"> Main article: <a href="http://en.wikipedia.org/wiki/History_of_polymerase_chain_reaction" title="History of polymerase chain reaction">History of polymerase chain reaction</a></div> A 1971 paper in the <a href="http://en.wikipedia.org/wiki/Journal_of_Molecular_Biology" title="Journal of Molecular Biology">Journal of Molecular Biology</a> by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers <i>in vitro</i>.<sup class="reference" id="cite_ref-52"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-52">[52]</a></sup> However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to <a href="http://en.wikipedia.org/wiki/Kary_Mullis" title="Kary Mullis">Kary Mullis</a>.<sup class="reference" id="cite_ref-53"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-53">[53]</a></sup><br /> <div class="thumb tright"> <div class="thumbinner" style="width: 222px;"> <a class="image" href="http://en.wikipedia.org/wiki/File:Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29,_c_1986._%289663810586%29.jpg"><img alt="" class="thumbimage" data-file-height="806" data-file-width="1250" height="142" src="http://upload.wikimedia.org/wikipedia/commons/thumb/d/db/Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29%2C_c_1986._%289663810586%29.jpg/220px-Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29%2C_c_1986._%289663810586%29.jpg" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/d/db/Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29%2C_c_1986._%289663810586%29.jpg/330px-Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29%2C_c_1986._%289663810586%29.jpg 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/d/db/Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29%2C_c_1986._%289663810586%29.jpg/440px-Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29%2C_c_1986._%289663810586%29.jpg 2x" width="220" /></a> <div class="thumbcaption"> <div class="magnify"> <a class="internal" href="http://en.wikipedia.org/wiki/File:Baby_Blue_-_a_prototype_polymerase_chain_reaction_%28PCR%29,_c_1986._%289663810586%29.jpg" title="Enlarge"><img alt="" height="11" src="http://bits.wikimedia.org/static-1.24wmf12/skins/common/images/magnify-clip.png" width="15" /></a></div> "Baby Blue", a 1986 prototype machine for doing PCR</div> </div> </div> When Mullis developed the PCR in 1983, he was working in <a href="http://en.wikipedia.org/wiki/Emeryville,_California" title="Emeryville, California">Emeryville</a>, California for <a href="http://en.wikipedia.org/wiki/Cetus_Corporation" title="Cetus Corporation">Cetus Corporation</a>, one of the first <a href="http://en.wikipedia.org/wiki/Biotechnology" title="Biotechnology">biotechnology</a> companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the <a href="http://en.wikipedia.org/wiki/California_State_Route_1" title="California State Route 1">Pacific Coast Highway</a> one night in his car.<sup class="reference" id="cite_ref-Mullis_54-0"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Mullis-54">[54]</a></sup> He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In <i><a href="http://en.wikipedia.org/wiki/Scientific_American" title="Scientific American">Scientific American</a></i>, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."<sup class="reference" id="cite_ref-55"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-55">[55]</a></sup> He was awarded the <a href="http://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistry" title="Nobel Prize in Chemistry">Nobel Prize in Chemistry</a> in 1993 for his invention,<sup class="reference" id="cite_ref-Kary_Mullis_Nobel_Lecture_5-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Kary_Mullis_Nobel_Lecture-5">[5]</a></sup> seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle (see below).<br /> At the core of the PCR method is the use of a suitable <a href="http://en.wikipedia.org/wiki/DNA_polymerase" title="DNA polymerase">DNA polymerase</a> able to withstand the high temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the <a class="mw-redirect" href="http://en.wikipedia.org/wiki/DNA_double_helix" title="DNA double helix">DNA double helix</a> after each <a href="http://en.wikipedia.org/wiki/DNA_replication" title="DNA replication">replication</a> cycle. The DNA polymerases initially employed for <a href="http://en.wikipedia.org/wiki/In_vitro" title="In vitro">in vitro</a> experiments presaging PCR were unable to withstand these high temperatures.<sup class="reference" id="cite_ref-Saiki1_3-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Saiki1-3">[3]</a></sup> So the early procedures for DNA replication were very inefficient and time consuming, and required large amounts of DNA polymerase and continuous handling throughout the process.<br /> The discovery in 1976 of <a href="http://en.wikipedia.org/wiki/Taq_polymerase" title="Taq polymerase">Taq polymerase</a> — a DNA polymerase purified from the <a href="http://en.wikipedia.org/wiki/Thermophile" title="Thermophile">thermophilic bacterium</a>, <i><a href="http://en.wikipedia.org/wiki/Thermus_aquaticus" title="Thermus aquaticus">Thermus aquaticus</a></i>, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments<sup class="reference" id="cite_ref-Chien_et_al._12-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Chien_et_al.-12">[12]</a></sup> such as hot springs — paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from <i>T. aquaticus</i> is stable at high temperatures remaining active even after DNA denaturation,<sup class="reference" id="cite_ref-Lawyer_et_al._13-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Lawyer_et_al.-13">[13]</a></sup> thus obviating the need to add new DNA polymerase after each cycle.<sup class="reference" id="cite_ref-Saiki2_4-1"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-Saiki2-4">[4]</a></sup> This allowed an automated thermocycler-based process for DNA amplification.<br /> <h3> <span class="mw-headline" id="Patent_disputes">Patent disputes</span></h3> The PCR technique was patented by Kary Mullis and assigned to <a href="http://en.wikipedia.org/wiki/Cetus_Corporation" title="Cetus Corporation">Cetus Corporation</a>, where Mullis worked when he invented the technique in 1983. The <i>Taq</i> polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by <a href="http://en.wikipedia.org/wiki/DuPont" title="DuPont">DuPont</a>. The pharmaceutical company <a href="http://en.wikipedia.org/wiki/Hoffmann-La_Roche" title="Hoffmann-La Roche">Hoffmann-La Roche</a> purchased the rights to the patents in 1992 and currently holds those that are still protected.<br /> A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and <a href="http://en.wikipedia.org/wiki/Promega" title="Promega">Promega</a>. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on March 28, 2005.<sup class="reference" id="cite_ref-56"><a href="http://en.wikipedia.org/wiki/Polymerase_chain_reaction#cite_note-56">[56]</a></sup><br /> <h2> <span class="mw-headline" id="References">References</span></h2> <div class="reflist references-column-width" style="-moz-column-width: 30em; -webkit-column-width: 30em; column-width: 30em; list-style-type: decimal;"> <ol class="references"> <li id="cite_note-Bartlett_.26_Stirling-1"> <span class="reference-text"><span class="citation journal">Bartlett, J. M. S.; Stirling, D. (2003). "A Short History of the Polymerase Chain Reaction". <i>PCR Protocols</i> <b>226</b>. pp. 3–6. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1385%2F1-59259-384-4%3A3" rel="nofollow">10.1385/1-59259-384-4:3</a>. <a href="http://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="http://en.wikipedia.org/wiki/Special:BookSources/1-59259-384-4" title="Special:BookSources/1-59259-384-4">1-59259-384-4</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=PCR+Protocols&rft.au=Bartlett%2C+J.+M.+S.&rft.aufirst=J.+M.+S.&rft.aulast=Bartlett&rft.au=Stirling%2C+D.&rft.btitle=A+Short+History+of+the+Polymerase+Chain+Reaction&rft.date=2003&rft.genre=bookitem&rft_id=info%3Adoi%2F10.1385%2F1-59259-384-4%3A3&rft.isbn=1-59259-384-4&rft.pages=3-6&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.volume=226"></span> <span class="plainlinks noprint" style="font-size: smaller;"><a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_doi/10.1385.2F1-59259-384-4:3&action=edit&editintro=Template:Cite_doi/editintro2">edit</a></span></span></li> <li id="cite_note-pat_mar-2"> <span class="reference-text"><a class="external text" href="http://www.google.co.jp/patents/US4683195" rel="nofollow">US4683195</a></span></li> <li id="cite_note-Saiki1-3"> <span class="reference-text"><span class="citation journal">Saiki, R.; Scharf, S.; Faloona, F.; Mullis, K.; Horn, G.; Erlich, H.; Arnheim, N. (1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". <i>Science</i> <b>230</b> (4732): 1350–1354. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1126%2Fscience.2999980" rel="nofollow">10.1126/science.2999980</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2999980" rel="nofollow">2999980</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Enzymatic+amplification+of+beta-globin+genomic+sequences+and+restriction+site+analysis+for+diagnosis+of+sickle+cell+anemia&rft.au=Arnheim%2C+N.&rft.au=Erlich%2C+H.&rft.au=Faloona%2C+F.&rft.aufirst=R.&rft.au=Horn%2C+G.&rft.aulast=Saiki&rft.au=Mullis%2C+K.&rft.au=Saiki%2C+R.&rft.au=Scharf%2C+S.&rft.date=1985&rft.genre=article&rft_id=info%3Adoi%2F10.1126%2Fscience.2999980&rft_id=info%3Apmid%2F2999980&rft.issue=4732&rft.jtitle=Science&rft.pages=1350-1354&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=230"></span><span class="plainlinks noprint" style="font-size: smaller;"> <a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_pmid/2999980&action=edit&editintro=Template:Cite_pmid/editintro2">edit</a></span></span></li> <li id="cite_note-Saiki2-4"> <span class="reference-text"><span class="citation journal">Saiki, R.; Gelfand, D.; Stoffel, S.; Scharf, S.; Higuchi, R.; Horn, G.; Mullis, K.; Erlich, H. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". <i>Science</i> <b>239</b> (4839): 487–491. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1126%2Fscience.2448875" rel="nofollow">10.1126/science.2448875</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2448875" rel="nofollow">2448875</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Primer-directed+enzymatic+amplification+of+DNA+with+a+thermostable+DNA+polymerase&rft.au=Erlich%2C+H.&rft.aufirst=R.&rft.au=Gelfand%2C+D.&rft.au=Higuchi%2C+R.&rft.au=Horn%2C+G.&rft.aulast=Saiki&rft.au=Mullis%2C+K.&rft.au=Saiki%2C+R.&rft.au=Scharf%2C+S.&rft.au=Stoffel%2C+S.&rft.date=1988&rft.genre=article&rft_id=info%3Adoi%2F10.1126%2Fscience.2448875&rft_id=info%3Apmid%2F2448875&rft.issue=4839&rft.jtitle=Science&rft.pages=487-491&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=239"></span><span class="plainlinks noprint" style="font-size: smaller;"> <a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_pmid/2448875&action=edit&editintro=Template:Cite_pmid/editintro2">edit</a></span></span></li> <li id="cite_note-Kary_Mullis_Nobel_Lecture-5"> <span class="reference-text"><a class="external text" href="http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html" rel="nofollow">Kary Mullis Nobel Lecture, December 8, 1993</a></span></li> <li id="cite_note-6"> <span class="reference-text"><span class="citation journal">Cheng, S.; Fockler, C.; Barnes, W. M.; Higuchi, R. (1994). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44063" rel="nofollow">"Effective Amplification of Long Targets from Cloned Inserts and Human Genomic DNA"</a>. <i>Proceedings of the National Academy of Sciences</i> <b>91</b> (12): 5695–5699. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1073%2Fpnas.91.12.5695" rel="nofollow">10.1073/pnas.91.12.5695</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44063" rel="nofollow">44063</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/8202550" rel="nofollow">8202550</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Effective+Amplification+of+Long+Targets+from+Cloned+Inserts+and+Human+Genomic+DNA&rft.au=Barnes%2C+W.+M.&rft.au=Cheng%2C+S.&rft.aufirst=S.&rft.au=Fockler%2C+C.&rft.au=Higuchi%2C+R.&rft.aulast=Cheng&rft.date=1994&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC44063&rft_id=info%3Adoi%2F10.1073%2Fpnas.91.12.5695&rft_id=info%3Apmc%2F44063&rft_id=info%3Apmid%2F8202550&rft.issue=12&rft.jtitle=Proceedings+of+the+National+Academy+of+Sciences&rft.pages=5695-5699&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=91"></span> <span class="plainlinks noprint" style="font-size: smaller;"><a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_doi/10.1073.2Fpnas.91.12.5695&action=edit&editintro=Template:Cite_doi/editintro2">edit</a></span></span></li> <li id="cite_note-7"> <span class="reference-text"><span class="citation journal">Carr AC, Moore SD (2012). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365123" rel="nofollow">"Robust quantification of polymerase chain reactions using global fitting"</a>. In Lucia, Alejandro. <i>PloS ONE</i> <b>7</b> (5): e37640. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1371%2Fjournal.pone.0037640" rel="nofollow">10.1371/journal.pone.0037640</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365123" rel="nofollow">3365123</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/22701526" rel="nofollow">22701526</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Robust+quantification+of+polymerase+chain+reactions+using+global+fitting&rft.au=Carr+AC%2C+Moore+SD&rft.aulast=Carr+AC%2C+Moore+SD&rft.date=2012&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC3365123&rft_id=info%3Adoi%2F10.1371%2Fjournal.pone.0037640&rft_id=info%3Apmc%2F3365123&rft_id=info%3Apmid%2F22701526&rft.issue=5&rft.jtitle=PloS+ONE&rft.pages=e37640&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=7"></span></span></li> <li id="cite_note-molecular_cloning-8"> <span class="reference-text"><span class="citation book">Joseph Sambrook and David W. Russel (2001). <i>Molecular Cloning: A Laboratory Manual</i> (3rd ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. <a href="http://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="http://en.wikipedia.org/wiki/Special:BookSources/0-879-69576-5" title="Special:BookSources/0-879-69576-5">0-879-69576-5</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.au=Joseph+Sambrook+and+David+W.+Russel&rft.aulast=Joseph+Sambrook+and+David+W.+Russel&rft.btitle=Molecular+Cloning%3A+A+Laboratory+Manual&rft.date=2001&rft.edition=3rd&rft.genre=book&rft.isbn=0-879-69576-5&rft.place=Cold+Spring+Harbor%2C+N.Y.&rft.pub=Cold+Spring+Harbor+Laboratory+Press&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span> Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction</span></li> <li id="cite_note-9"> <span class="reference-text"><span class="citation journal">Pavlov, A. R.; Pavlova, N. V.; Kozyavkin, S. A.; Slesarev, A. I. (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications☆". <i>Trends in Biotechnology</i> <b>22</b> (5): 253–260. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2Fj.tibtech.2004.02.011" rel="nofollow">10.1016/j.tibtech.2004.02.011</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/15109812" rel="nofollow">15109812</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Recent+developments+in+the+optimization+of+thermostable+DNA+polymerases+for+efficient+applications%E2%98%86&rft.aufirst=A.+R.&rft.au=Kozyavkin%2C+S.+A.&rft.aulast=Pavlov&rft.au=Pavlov%2C+A.+R.&rft.au=Pavlova%2C+N.+V.&rft.au=Slesarev%2C+A.+I.&rft.date=2004&rft.genre=article&rft_id=info%3Adoi%2F10.1016%2Fj.tibtech.2004.02.011&rft_id=info%3Apmid%2F15109812&rft.issue=5&rft.jtitle=Trends+in+Biotechnology&rft.pages=253-260&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=22"></span> <span class="plainlinks noprint" style="font-size: smaller;"><a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_doi/10.1016.2Fj.tibtech.2004.02.011&action=edit&editintro=Template:Cite_doi/editintro2">edit</a></span></span></li> <li id="cite_note-10"> <span class="reference-text"><span class="citation journal">Rychlik W, Spencer WJ, Rhoads RE (1990). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332522" rel="nofollow">"Optimization of the annealing temperature for DNA amplification in vitro"</a>. <i>Nucl Acids Res</i> <b>18</b> (21): 6409–6412. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1093%2Fnar%2F18.21.6409" rel="nofollow">10.1093/nar/18.21.6409</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332522" rel="nofollow">332522</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2243783" rel="nofollow">2243783</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Optimization+of+the+annealing+temperature+for+DNA+amplification+in+vitro&rft.aulast=Rychlik+W%2C+Spencer+WJ%2C+Rhoads+RE&rft.au=Rychlik+W%2C+Spencer+WJ%2C+Rhoads+RE&rft.date=1990&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC332522&rft_id=info%3Adoi%2F10.1093%2Fnar%2F18.21.6409&rft_id=info%3Apmc%2F332522&rft_id=info%3Apmid%2F2243783&rft.issue=21&rft.jtitle=Nucl+Acids+Res&rft.pages=6409-6412&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=18"></span></span></li> <li id="cite_note-antibody_hot_start-11"> <span class="reference-text"><span class="citation journal">Sharkey, D. J.; Scalice, E. R.; Christy, K. G.; Atwood, S. M.; Daiss, J. L. (1994). "Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction". <i>Bio/Technology</i> <b>12</b> (5): 506–509. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1038%2Fnbt0594-506" rel="nofollow">10.1038/nbt0594-506</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Antibodies+as+Thermolabile+Switches%3A+High+Temperature+Triggering+for+the+Polymerase+Chain+Reaction&rft.au=Atwood%2C+S.+M.&rft.au=Christy%2C+K.+G.&rft.au=Daiss%2C+J.+L.&rft.aufirst=D.+J.&rft.aulast=Sharkey&rft.au=Scalice%2C+E.+R.&rft.au=Sharkey%2C+D.+J.&rft.date=1994&rft.genre=article&rft_id=info%3Adoi%2F10.1038%2Fnbt0594-506&rft.issue=5&rft.jtitle=Bio%2FTechnology&rft.pages=506-509&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=12"></span> <span class="plainlinks noprint" style="font-size: smaller;"><a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_doi/10.1038.2Fnbt0594-506&action=edit&editintro=Template:Cite_doi/editintro2">edit</a></span></span></li> <li id="cite_note-Chien_et_al.-12"> <span class="reference-text"><span class="citation journal">Chien A, Edgar DB, Trela JM (1976). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC232952" rel="nofollow">"Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus"</a>. <i>J. Bacteriol</i> <b>127</b> (3): 1550–1557. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC232952" rel="nofollow">232952</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/8432" rel="nofollow">8432</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Deoxyribonucleic+acid+polymerase+from+the+extreme+thermophile+Thermus+aquaticus&rft.au=Chien+A%2C+Edgar+DB%2C+Trela+JM&rft.aulast=Chien+A%2C+Edgar+DB%2C+Trela+JM&rft.date=1976&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC232952&rft_id=info%3Apmc%2F232952&rft_id=info%3Apmid%2F8432&rft.issue=3&rft.jtitle=J.+Bacteriol&rft.pages=1550-1557&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=127"></span></span></li> <li id="cite_note-Lawyer_et_al.-13"> <span class="reference-text"><span class="citation journal">Lawyer, F.; Stoffel, S.; Saiki, R.; Chang, S.; Landre, P.; Abramson, R.; Gelfand, D. (1993). "High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity". <i>PCR methods and applications</i> <b>2</b> (4): 275–287. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/8324500" rel="nofollow">8324500</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=High-level+expression%2C+purification%2C+and+enzymatic+characterization+of+full-length+Thermus+aquaticus+DNA+polymerase+and+a+truncated+form+deficient+in+5%27+to+3%27+exonuclease+activity&rft.au=Abramson%2C+R.&rft.au=Chang%2C+S.&rft.aufirst=F.&rft.au=Gelfand%2C+D.&rft.au=Landre%2C+P.&rft.aulast=Lawyer&rft.au=Lawyer%2C+F.&rft.au=Saiki%2C+R.&rft.au=Stoffel%2C+S.&rft.date=1993&rft.genre=article&rft_id=info%3Apmid%2F8324500&rft.issue=4&rft.jtitle=PCR+methods+and+applications&rft.pages=275-287&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=2"></span><span class="plainlinks noprint" style="font-size: smaller;"> <a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_pmid/8324500&action=edit&editintro=Template:Cite_pmid/editintro2">edit</a></span></span></li> <li id="cite_note-14"> <span class="reference-text">Campbell Biology,7th edition</span></li> <li id="cite_note-15"> <span class="reference-text">PCR from problematic templates. Focus 22:1 p.10 (2000).</span></li> <li id="cite_note-16"> <span class="reference-text">Helpful tips for PCR. Focus 22:1 p.12 (2000).</span></li> <li id="cite_note-17"> <span class="reference-text"><span class="citation journal">Sarkar, G.; Kapelner, S.; Sommer, S. (1990). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332902" rel="nofollow">"Formamide can dramatically improve the specificity of PCR"</a>. <i>Nucleic Acids Research</i> <b>18</b> (24): 7465. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1093%2Fnar%2F18.24.7465" rel="nofollow">10.1093/nar/18.24.7465</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332902" rel="nofollow">332902</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2259646" rel="nofollow">2259646</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Formamide+can+dramatically+improve+the+specificity+of+PCR&rft.aufirst=G.&rft.au=Kapelner%2C+S.&rft.aulast=Sarkar&rft.au=Sarkar%2C+G.&rft.au=Sommer%2C+S.&rft.date=1990&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC332902&rft_id=info%3Adoi%2F10.1093%2Fnar%2F18.24.7465&rft_id=info%3Apmc%2F332902&rft_id=info%3Apmid%2F2259646&rft.issue=24&rft.jtitle=Nucleic+Acids+Research&rft.pages=7465&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=18"></span><span class="plainlinks noprint" style="font-size: smaller;"> <a class="external text" href="http://en.wikipedia.org/w/index.php?title=Template:Cite_pmid/2259646&action=edit&editintro=Template:Cite_pmid/editintro2">edit</a></span></span></li> <li id="cite_note-ePCR-18"> <span class="reference-text"><span class="citation web"><a class="external text" href="http://www.ncbi.nlm.nih.gov/sutils/e-pcr/" rel="nofollow">"Electronic PCR"</a>. NCBI – National Center for Biotechnology Information<span class="reference-accessdate">. Retrieved 13 March 2012</span>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.btitle=Electronic+PCR&rft.genre=book&rft_id=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fsutils%2Fe-pcr%2F&rft.pub=NCBI+%E2%80%93+National+Center+for+Biotechnology+Information&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-Hybrid-19"> <span class="reference-text"><span class="citation book">Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes". In Kieleczawa J. <i>DNA Sequencing II: Optimizing Preparation and Cleanup</i>. Jones and Bartlett. pp. 241–257. <a href="http://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="http://en.wikipedia.org/wiki/Special:BookSources/0-7637-3383-0" title="Special:BookSources/0-7637-3383-0">0-7637-3383-0</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=DNA+Sequencing+II%3A+Optimizing+Preparation+and+Cleanup&rft.aulast=Pavlov+AR%2C+Pavlova+NV%2C+Kozyavkin+SA%2C+Slesarev+AI&rft.au=Pavlov+AR%2C+Pavlova+NV%2C+Kozyavkin+SA%2C+Slesarev+AI&rft.btitle=Thermostable+DNA+Polymerases+for+a+Wide+Spectrum+of+Applications%3A+Comparison+of+a+Robust+Hybrid+TopoTaq+to+other+enzymes&rft.date=2006&rft.genre=bookitem&rft.isbn=0-7637-3383-0&rft.pages=241-257&rft.pub=Jones+and+Bartlett&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-20"> <span class="reference-text"><span class="citation web"><a class="external text" href="http://photoscience.la.asu.edu/photosyn/courses/BIO_343/lecture/DNAtech.html" rel="nofollow">"Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343)"</a>. Arizona State University<span class="reference-accessdate">. Retrieved 2007-10-29</span>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.btitle=Chemical+Synthesis%2C+Sequencing%2C+and+Amplification+of+DNA+%28class+notes+on+MBB%2FBIO+343%29&rft.genre=book&rft_id=http%3A%2F%2Fphotoscience.la.asu.edu%2Fphotosyn%2Fcourses%2FBIO_343%2Flecture%2FDNAtech.html&rft.pub=Arizona+State+University&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-Cai2014-21"> <span class="reference-text"><span class="citation journal">Cai, H; Caswell JL, Prescott JF (March 2014). <a class="external text" href="http://vet.sagepub.com/content/51/2/341.full" rel="nofollow">"Nonculture Molecular Techniques for Diagnosis of Bacterial Disease in Animals: A Diagnostic Laboratory Perspective"</a>. <i>Veterinary Pathology</i> <b>51</b> (2): 341–350. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1177%2F0300985813511132" rel="nofollow">10.1177/0300985813511132</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/24569613" rel="nofollow">24569613</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Nonculture+Molecular+Techniques+for+Diagnosis+of+Bacterial+Disease+in+Animals%3A+A+Diagnostic+Laboratory+Perspective&rft.au=Cai%2C+H&rft.aufirst=H&rft.aulast=Cai&rft.date=March+2014&rft.genre=article&rft_id=http%3A%2F%2Fvet.sagepub.com%2Fcontent%2F51%2F2%2F341.full&rft_id=info%3Adoi%2F10.1177%2F0300985813511132&rft_id=info%3Apmid%2F24569613&rft.issue=2&rft.jtitle=Veterinary+Pathology&rft.pages=341-350&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=51"></span> </span></li> <li id="cite_note-22"> <span class="reference-text"><span class="citation book">Salis AD (2009). "Applications in Clinical Microbiology". <i>Real-Time PCR: Current Technology and Applications</i>. Caister Academic Press. <a href="http://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="http://en.wikipedia.org/wiki/Special:BookSources/978-1-904455-39-4" title="Special:BookSources/978-1-904455-39-4">978-1-904455-39-4</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Real-Time+PCR%3A+Current+Technology+and+Applications&rft.aulast=Salis+AD&rft.au=Salis+AD&rft.btitle=Applications+in+Clinical+Microbiology&rft.date=2009&rft.genre=bookitem&rft.isbn=978-1-904455-39-4&rft.pub=Caister+Academic+Press&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-23"> <span class="reference-text"><span class="citation journal">Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, and Markham AF (1989). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC317639" rel="nofollow">"Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)"</a>. <i>Nucleic Acids Research</i> <b>17</b> (7): 2503–2516. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1093%2Fnar%2F17.7.2503" rel="nofollow">10.1093/nar/17.7.2503</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC317639" rel="nofollow">317639</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2785681" rel="nofollow">2785681</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Analysis+of+any+point+mutation+in+DNA.+The+amplification+refractory+mutation+system+%28ARMS%29&rft.aulast=Newton+CR%2C+Graham+A%2C+Heptinstall+LE%2C+Powell+SJ%2C+Summers+C%2C+Kalsheker+N%2C+Smith+JC%2C+and+Markham+AF&rft.au=Newton+CR%2C+Graham+A%2C+Heptinstall+LE%2C+Powell+SJ%2C+Summers+C%2C+Kalsheker+N%2C+Smith+JC%2C+and+Markham+AF&rft.date=1989&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC317639&rft_id=info%3Adoi%2F10.1093%2Fnar%2F17.7.2503&rft_id=info%3Apmc%2F317639&rft_id=info%3Apmid%2F2785681&rft.issue=7&rft.jtitle=Nucleic+Acids+Research&rft.pages=2503-2516&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=17"></span></span></li> <li id="cite_note-Stemmer_et_al.-24"> <span class="reference-text"><span class="citation journal">Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". <i>Gene</i> <b>164</b> (1): 49–53. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2F0378-1119%2895%2900511-4" rel="nofollow">10.1016/0378-1119(95)00511-4</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/7590320" rel="nofollow">7590320</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Single-step+assembly+of+a+gene+and+entire+plasmid+from+large+numbers+of+oligodeoxyribonucleotides&rft.aulast=Stemmer+WP%2C+Crameri+A%2C+Ha+KD%2C+Brennan+TM%2C+Heyneker+HL&rft.au=Stemmer+WP%2C+Crameri+A%2C+Ha+KD%2C+Brennan+TM%2C+Heyneker+HL&rft.date=1995&rft.genre=article&rft_id=info%3Adoi%2F10.1016%2F0378-1119%2895%2900511-4&rft_id=info%3Apmid%2F7590320&rft.issue=1&rft.jtitle=Gene&rft.pages=49-53&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=164"></span></span></li> <li id="cite_note-Innis_et_al.-25"> <span class="reference-text"><span class="citation journal">Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC282767" rel="nofollow">"DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA"</a>. <i>Proc Natl Acad Sci USA</i> <b>85</b> (24): 9436–4940. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1073%2Fpnas.85.24.9436" rel="nofollow">10.1073/pnas.85.24.9436</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC282767" rel="nofollow">282767</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/3200828" rel="nofollow">3200828</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=DNA+sequencing+with+Thermus+aquaticus+DNA+polymerase+and+direct+sequencing+of+polymerase+chain+reaction-amplified+DNA&rft.au=Innis+MA%2C+Myambo+KB%2C+Gelfand+DH%2C+Brow+MA.&rft.aulast=Innis+MA%2C+Myambo+KB%2C+Gelfand+DH%2C+Brow+MA.&rft.date=1988&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC282767&rft_id=info%3Adoi%2F10.1073%2Fpnas.85.24.9436&rft_id=info%3Apmc%2F282767&rft_id=info%3Apmid%2F3200828&rft.issue=24&rft.jtitle=Proc+Natl+Acad+Sci+USA&rft.pages=9436-4940&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=85"></span></span></li> <li id="cite_note-Pierce_and_Wangh-26"> <span class="reference-text"><span class="citation journal">Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". <i>Methods Mol Med</i>. Methods in Molecular Medicine™ <b>132</b>: 65–85. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1007%2F978-1-59745-298-4_7" rel="nofollow">10.1007/978-1-59745-298-4_7</a>. <a href="http://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="http://en.wikipedia.org/wiki/Special:BookSources/978-1-58829-578-1" title="Special:BookSources/978-1-58829-578-1">978-1-58829-578-1</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/17876077" rel="nofollow">17876077</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Linear-after-the-exponential+polymerase+chain+reaction+and+allied+technologies+Real-time+detection+strategies+for+rapid%2C+reliable+diagnosis+from+single+cells&rft.aulast=Pierce+KE+and+Wangh+LJ&rft.au=Pierce+KE+and+Wangh+LJ&rft.date=2007&rft.genre=article&rft_id=info%3Adoi%2F10.1007%2F978-1-59745-298-4_7&rft_id=info%3Apmid%2F17876077&rft.isbn=978-1-58829-578-1&rft.jtitle=Methods+Mol+Med.&rft.pages=65-85&rft.series=Methods+in+Molecular+Medicine%E2%84%A2&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=132"></span></span></li> <li id="cite_note-Schwartz_et_al.-27"> <span class="reference-text"><span class="citation journal">Schwartz JJ, Lee C, Shendure J. (2012). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433648" rel="nofollow">"Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules"</a>. <i>Nature Methods</i> <b>9</b> (9): 913–915. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1038%2Fnmeth.2137" rel="nofollow">10.1038/nmeth.2137</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433648" rel="nofollow">3433648</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/22886093" rel="nofollow">22886093</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Accurate+gene+synthesis+with+tag-directed+retrieval+of+sequence-verified+DNA+molecules&rft.aulast=Schwartz+JJ%2C+Lee+C%2C+Shendure+J.&rft.au=Schwartz+JJ%2C+Lee+C%2C+Shendure+J.&rft.date=2012&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC3433648&rft_id=info%3Adoi%2F10.1038%2Fnmeth.2137&rft_id=info%3Apmc%2F3433648&rft_id=info%3Apmid%2F22886093&rft.issue=9&rft.jtitle=Nature+Methods&rft.pages=913-915&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=9"></span></span></li> <li id="cite_note-28"> <span class="reference-text"><span class="citation journal">Vincent,Myriam, Xu, Yan, and Kong, Huimin (2004). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1249482" rel="nofollow">"Helicase-dependent isothermal DNA amplification"</a>. <i>EMBO Reports</i> <b>5</b> (8): 795–800. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1038%2Fsj.embor.7400200" rel="nofollow">10.1038/sj.embor.7400200</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1249482" rel="nofollow">1249482</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/15247927" rel="nofollow">15247927</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Helicase-dependent+isothermal+DNA+amplification&rft.aulast=Vincent%2CMyriam%2C+Xu%2C+Yan%2C+and+Kong%2C+Huimin&rft.au=Vincent%2CMyriam%2C+Xu%2C+Yan%2C+and+Kong%2C+Huimin&rft.date=2004&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC1249482&rft_id=info%3Adoi%2F10.1038%2Fsj.embor.7400200&rft_id=info%3Apmc%2F1249482&rft_id=info%3Apmid%2F15247927&rft.issue=8&rft.jtitle=EMBO+Reports&rft.pages=795-800&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=5"></span></span></li> <li id="cite_note-general_hot_start-29"> <span class="reference-text"><span class="citation journal">Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC312262" rel="nofollow">"Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications"</a>. <i>Nucleic Acids Research</i> <b>20</b> (7): 1717–1723. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1093%2Fnar%2F20.7.1717" rel="nofollow">10.1093/nar/20.7.1717</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC312262" rel="nofollow">312262</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/1579465" rel="nofollow">1579465</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Prevention+of+pre-PCR+mis-priming+and+primer+dimerization+improves+low-copy-number+amplifications&rft.aulast=Q.+Chou%2C+M.+Russell%2C+D.E.+Birch%2C+J.+Raymond+and+W.+Bloch&rft.au=Q.+Chou%2C+M.+Russell%2C+D.E.+Birch%2C+J.+Raymond+and+W.+Bloch&rft.date=1992&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC312262&rft_id=info%3Adoi%2F10.1093%2Fnar%2F20.7.1717&rft_id=info%3Apmc%2F312262&rft_id=info%3Apmid%2F1579465&rft.issue=7&rft.jtitle=Nucleic+Acids+Research&rft.pages=1717-1723&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=20"></span></span></li> <li id="cite_note-antibody_hot_start_2-30"> <span class="reference-text">Kellogg D E, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert P D, Chenchik A. 1994. TaqStart Antibody: hot start PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16 (6):1134-1137<span class="citation journal">Kellogg, DE; Rybalkin, I; Chen, S; Mukhamedova, N; Vlasik, T; Siebert, PD; Chenchik, A (1994). "TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase". <i>BioTechniques</i> <b>16</b> (6): 1134–7. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/8074881" rel="nofollow">8074881</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=TaqStart+Antibody%3A+%22hot+start%22+PCR+facilitated+by+a+neutralizing+monoclonal+antibody+directed+against+Taq+DNA+polymerase&rft.au=Chen%2C+S&rft.au=Chenchik%2C+A&rft.aufirst=DE&rft.au=Kellogg%2C+DE&rft.aulast=Kellogg&rft.au=Mukhamedova%2C+N&rft.au=Rybalkin%2C+I&rft.au=Siebert%2C+PD&rft.au=Vlasik%2C+T&rft.date=1994&rft.genre=article&rft_id=info%3Apmid%2F8074881&rft.issue=6&rft.jtitle=BioTechniques&rft.pages=1134-7&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=16"></span></span></li> <li id="cite_note-31"> <span class="reference-text"><span class="citation journal">San Millan RM, Martinez-Ballesteros I, Rementeria A, Garaizar J, Bikandi J (2013). "Online exercise for the design and simulation of PCR and PCR-RFLP experiments". <i>BMC Research Notes</i> <b>6</b>: 513. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1186%2F1756-0500-6-513" rel="nofollow">10.1186/1756-0500-6-513</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/24314313" rel="nofollow">24314313</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Online+exercise+for+the+design+and+simulation+of+PCR+and+PCR-RFLP+experiments&rft.aulast=San+Millan+RM%2C+Martinez-Ballesteros+I%2C+Rementeria+A%2C+Garaizar+J%2C+Bikandi+J&rft.au=San+Millan+RM%2C+Martinez-Ballesteros+I%2C+Rementeria+A%2C+Garaizar+J%2C+Bikandi+J&rft.date=2013&rft.genre=article&rft_id=info%3Adoi%2F10.1186%2F1756-0500-6-513&rft_id=info%3Apmid%2F24314313&rft.jtitle=BMC+Research+Notes&rft.pages=513&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=6"></span></span></li> <li id="cite_note-32"> <span class="reference-text"><span class="citation journal">E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". <i>Genomics</i> <b>20</b> (2): 176–83. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1006%2Fgeno.1994.1151" rel="nofollow">10.1006/geno.1994.1151</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/8020964" rel="nofollow">8020964</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Genome+fingerprinting+by+simple+sequence+repeat+%28SSR%29-anchored+polymerase+chain+reaction+amplification&rft.au=E.+Zietkiewicz%2C+A.+Rafalski%2C+and+D.+Labuda&rft.aulast=E.+Zietkiewicz%2C+A.+Rafalski%2C+and+D.+Labuda&rft.date=1994&rft.genre=article&rft_id=info%3Adoi%2F10.1006%2Fgeno.1994.1151&rft_id=info%3Apmid%2F8020964&rft.issue=2&rft.jtitle=Genomics&rft.pages=176-83&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=20"></span></span></li> <li id="cite_note-Ochman_et_al.-33"> <span class="reference-text"><span class="citation journal">Ochman H, Gerber AS, Hartl DL (1988). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1203539" rel="nofollow">"Genetic Applications of an Inverse Polymerase Chain Reaction"</a>. <i>Genetics</i> <b>120</b> (3): 621–623. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1203539" rel="nofollow">1203539</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2852134" rel="nofollow">2852134</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Genetic+Applications+of+an+Inverse+Polymerase+Chain+Reaction&rft.aulast=Ochman+H%2C+Gerber+AS%2C+Hartl+DL&rft.au=Ochman+H%2C+Gerber+AS%2C+Hartl+DL&rft.date=1988&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC1203539&rft_id=info%3Apmc%2F1203539&rft_id=info%3Apmid%2F2852134&rft.issue=3&rft.jtitle=Genetics&rft.pages=621-623&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=120"></span></span></li> <li id="cite_note-Mueller_and_Wold-34"> <span class="reference-text"><span class="citation journal">Mueller PR, Wold B (1988). "<i>In vivo</i> footprinting of a muscle specific enhancer by ligation mediated PCR". <i>Science</i> <b>246</b> (4931): 780–786. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1126%2Fscience.2814500" rel="nofollow">10.1126/science.2814500</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2814500" rel="nofollow">2814500</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=%27%27In+vivo%27%27+footprinting+of+a+muscle+specific+enhancer+by+ligation+mediated+PCR&rft.aulast=Mueller+PR%2C+Wold+B&rft.au=Mueller+PR%2C+Wold+B&rft.date=1988&rft.genre=article&rft_id=info%3Adoi%2F10.1126%2Fscience.2814500&rft_id=info%3Apmid%2F2814500&rft.issue=4931&rft.jtitle=Science&rft.pages=780-786&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=246"></span></span></li> <li id="cite_note-35"> <span class="reference-text"><span class="citation journal">Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC38513" rel="nofollow">"Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands"</a>. <i>Proc Natl Acad Sci USA</i> <b>93</b> (13): 9821–9826. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1073%2Fpnas.93.18.9821" rel="nofollow">10.1073/pnas.93.18.9821</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC38513" rel="nofollow">38513</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/8790415" rel="nofollow">8790415</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Methylation-specific+PCR%3A+a+novel+PCR+assay+for+methylation+status+of+CpG+islands&rft.au=Herman+JG%2C+Graff+JR%2C+My%C3%B6h%C3%A4nen+S%2C+Nelkin+BD%2C+Baylin+SB&rft.aulast=Herman+JG%2C+Graff+JR%2C+My%C3%B6h%C3%A4nen+S%2C+Nelkin+BD%2C+Baylin+SB&rft.date=1996&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC38513&rft_id=info%3Adoi%2F10.1073%2Fpnas.93.18.9821&rft_id=info%3Apmc%2F38513&rft_id=info%3Apmid%2F8790415&rft.issue=13&rft.jtitle=Proc+Natl+Acad+Sci+USA&rft.pages=9821-9826&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=93"></span></span></li> <li id="cite_note-Isenbarger_.27.27et_al..27.27.-36"> <span class="reference-text"><span class="citation journal">Isenbarger TA, Finney M, Ríos-Velázquez C, Handelsman J, Ruvkun G (2008). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2227730" rel="nofollow">"Miniprimer PCR, a New Lens for Viewing the Microbial World"</a>. <i>Applied and Environmental Microbiology</i> <b>74</b> (3): 840–9. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1128%2FAEM.01933-07" rel="nofollow">10.1128/AEM.01933-07</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2227730" rel="nofollow">2227730</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/18083877" rel="nofollow">18083877</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Miniprimer+PCR%2C+a+New+Lens+for+Viewing+the+Microbial+World&rft.au=Isenbarger+TA%2C+Finney+M%2C+R%C3%ADos-Vel%C3%A1zquez+C%2C+Handelsman+J%2C+Ruvkun+G&rft.aulast=Isenbarger+TA%2C+Finney+M%2C+R%C3%ADos-Vel%C3%A1zquez+C%2C+Handelsman+J%2C+Ruvkun+G&rft.date=2008&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2227730&rft_id=info%3Adoi%2F10.1128%2FAEM.01933-07&rft_id=info%3Apmc%2F2227730&rft_id=info%3Apmid%2F18083877&rft.issue=3&rft.jtitle=Applied+and+Environmental+Microbiology&rft.pages=840-9&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=74"></span></span></li> <li id="cite_note-37"> <span class="reference-text"><span class="citation journal">Cenchao Shen, Wenjuan Yang, Qiaoli Ji, Hisaji Maki, Anjie Dong, Zhizhou Zhang (2009). "NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions". <i>Nanotechnology</i> <b>20</b>: 455103. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1088%2F0957-4484%2F20%2F45%2F455103" rel="nofollow">10.1088/0957-4484/20/45/455103</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=NanoPCR+observation%3A+different+levels+of+DNA+replication+fidelity+in+nanoparticle-enhanced+polymerase+chain+reactions&rft.au=Cenchao+Shen%2C+Wenjuan+Yang%2C+Qiaoli+Ji%2C+Hisaji+Maki%2C+Anjie+Dong%2C+Zhizhou+Zhang&rft.aulast=Cenchao+Shen%2C+Wenjuan+Yang%2C+Qiaoli+Ji%2C+Hisaji+Maki%2C+Anjie+Dong%2C+Zhizhou+Zhang&rft.date=2009&rft.genre=article&rft_id=info%3Adoi%2F10.1088%2F0957-4484%2F20%2F45%2F455103&rft.jtitle=Nanotechnology&rft.pages=455103&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=20"></span></span></li> <li id="cite_note-38"> <span class="reference-text"><span class="citation book">Shen, Cenchao (2013). <i>An Overview of Nanoparticle‐Assisted Polymerase Chain Reaction Technology</i>. US: Wiley-Blackwell Publishing Ltd. pp. 97–106.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.aufirst=Cenchao&rft.aulast=Shen&rft.au=Shen%2C+Cenchao&rft.btitle=An+Overview+of+Nanoparticle%E2%80%90Assisted+Polymerase+Chain+Reaction+Technology&rft.date=2013&rft.genre=book&rft.pages=97-106&rft.place=US&rft.pub=Wiley-Blackwell+Publishing+Ltd.&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-39"> <span class="reference-text"><span class="citation journal">Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989). "Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap exten-sion". <i>Gene</i> <b>77</b> (1): 61–68. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2F0378-1119%2889%2990359-4" rel="nofollow">10.1016/0378-1119(89)90359-4</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2744488" rel="nofollow">2744488</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Engineering+hybrid+genes+without+the+use+of+restriction+enzymes%3A+gene+splicing+by+overlap+exten-sion&rft.au=Horton+RM%2C+Hunt+HD%2C+Ho+SN%2C+Pullen+JK%2C+Pease+LR&rft.aulast=Horton+RM%2C+Hunt+HD%2C+Ho+SN%2C+Pullen+JK%2C+Pease+LR&rft.date=1989&rft.genre=article&rft_id=info%3Adoi%2F10.1016%2F0378-1119%2889%2990359-4&rft_id=info%3Apmid%2F2744488&rft.issue=1&rft.jtitle=Gene&rft.pages=61-68&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=77"></span></span></li> <li id="cite_note-40"> <span class="reference-text"><span class="citation book">Moller, Simon (2006). <i>PCR (THE BASICS)</i>. US: Taylor & Francis Group. p. 144.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.aufirst=Simon&rft.aulast=Moller&rft.au=Moller%2C+Simon&rft.btitle=PCR+%28THE+BASICS%29&rft.date=2006&rft.genre=book&rft.pages=144&rft.place=US&rft.pub=Taylor+%26+Francis+Group&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-41"> <span class="reference-text"><span class="citation journal">David, F. and Turlotte, E., (1998). "An Isothermal Amplification Method". <i>C.R.Acad. Sci Paris, Life Science</i> <b>321</b> (1): 909–914. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2FS0764-4469%2899%2980005-5" rel="nofollow">10.1016/S0764-4469(99)80005-5</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=An+Isothermal+Amplification+Method&rft.au=David%2C+F.+and+Turlotte%2C+E.%2C&rft.aulast=David%2C+F.+and+Turlotte%2C+E.%2C&rft.date=1998&rft.genre=article&rft_id=info%3Adoi%2F10.1016%2FS0764-4469%2899%2980005-5&rft.issue=1&rft.jtitle=C.R.Acad.+Sci+Paris%2C+Life+Science&rft.pages=909-914&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=321"></span></span></li> <li id="cite_note-42"> <span class="reference-text"><span class="citation web">Fabrice David (September–October 2002). <a class="external text" href="http://www.lab-rech-associatives.com/pdf/Utiliser%20la%20Topologie%20de%20l%27ADN.pdf" rel="nofollow">"Utiliser les propriétés topologiques de l’ADN: une nouvelle arme contre les agents pathogènes"</a> (PDF) <b>92</b>. Fusion.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.au=Fabrice+David&rft.aulast=Fabrice+David&rft.btitle=Utiliser+les+propri%C3%A9t%C3%A9s+topologiques+de+l%E2%80%99ADN%3A+une+nouvelle+arme+contre+les+agents+pathog%C3%A8nes&rft.date=September-October+2002&rft.genre=book&rft_id=http%3A%2F%2Fwww.lab-rech-associatives.com%2Fpdf%2FUtiliser%2520la%2520Topologie%2520de%2520l%27ADN.pdf&rft.pub=Fusion&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.volume=92"></span>(in French)</span></li> <li id="cite_note-43"> <span class="reference-text"><span class="citation journal">Bing, D. H., C. Boles, F. N. Rehman, M. Audeh, M. Belmarsh, B. Kelley, and C. P. Adams. (1996). <a class="external text" href="http://www.promega.com/geneticidproc/ussymp7proc/0726.html" rel="nofollow">"Bridge amplification: a solid phase PCR system for the amplification and detection of allelic differences in single copy genes"</a>. <i>Genetic Identity Conference Proceedings, Seventh International Symposium on Human Identification</i>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Bridge+amplification%3A+a+solid+phase+PCR+system+for+the+amplification+and+detection+of+allelic+differences+in+single+copy+genes&rft.au=Bing%2C+D.+H.%2C+C.+Boles%2C+F.+N.+Rehman%2C+M.+Audeh%2C+M.+Belmarsh%2C+B.+Kelley%2C+and+C.+P.+Adams.&rft.aulast=Bing%2C+D.+H.%2C+C.+Boles%2C+F.+N.+Rehman%2C+M.+Audeh%2C+M.+Belmarsh%2C+B.+Kelley%2C+and+C.+P.+Adams.&rft.date=1996&rft.genre=article&rft_id=http%3A%2F%2Fwww.promega.com%2Fgeneticidproc%2Fussymp7proc%2F0726.html&rft.jtitle=Genetic+Identity+Conference+Proceedings%2C+Seventh+International+Symposium+on+Human+Identification&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal"></span></span></li> <li id="cite_note-44"> <span class="reference-text"><span class="citation journal">Khan Z, Poetter K, Park DJ (2008). "Enhanced solid phase PCR: mechanisms to increase priming by solid support primers". <i>Analytical Biochemistry</i> <b>375</b> (2): 391–393. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2Fj.ab.2008.01.021" rel="nofollow">10.1016/j.ab.2008.01.021</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/18267099" rel="nofollow">18267099</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Enhanced+solid+phase+PCR%3A+mechanisms+to+increase+priming+by+solid+support+primers&rft.au=Khan+Z%2C+Poetter+K%2C+Park+DJ&rft.aulast=Khan+Z%2C+Poetter+K%2C+Park+DJ&rft.date=2008&rft.genre=article&rft_id=info%3Adoi%2F10.1016%2Fj.ab.2008.01.021&rft_id=info%3Apmid%2F18267099&rft.issue=2&rft.jtitle=Analytical+Biochemistry&rft.pages=391-393&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=375"></span></span></li> <li id="cite_note-45"> <span class="reference-text"><span class="citation journal">Raoult, D; G Aboudharam, E Crubezy, G Larrouy, B Ludes, M Drancourt (2000-11-07). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC18844" rel="nofollow">"Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death"</a>. <i>Proc. Natl. Acad. Sci. U.S.A.</i> <b>97</b> (23): 12800–12803. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1073%2Fpnas.220225197" rel="nofollow">10.1073/pnas.220225197</a>. <a href="http://en.wikipedia.org/wiki/International_Standard_Serial_Number" title="International Standard Serial Number">ISSN</a> <a class="external text" href="http://www.worldcat.org/issn/0027-8424" rel="nofollow">0027-8424</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC18844" rel="nofollow">18844</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/11058154" rel="nofollow">11058154</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Molecular+identification+by+%22suicide+PCR%22+of+Yersinia+pestis+as+the+agent+of+medieval+black+death&rft.aufirst=D&rft.aulast=Raoult&rft.au=Raoult%2C+D&rft.date=2000-11-07&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC18844&rft_id=info%3Adoi%2F10.1073%2Fpnas.220225197&rft_id=info%3Apmc%2F18844&rft_id=info%3Apmid%2F11058154&rft.issn=0027-8424&rft.issue=23&rft.jtitle=Proc.+Natl.+Acad.+Sci.+U.S.A.&rft.pages=12800-12803&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=97"></span> </span></li> <li id="cite_note-46"> <span class="reference-text"><span class="citation journal">Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". <i>Genomics</i> <b>25</b> (3): 674–81. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2F0888-7543%2895%2980010-J" rel="nofollow">10.1016/0888-7543(95)80010-J</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/7759102" rel="nofollow">7759102</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Thermal+asymmetric+interlaced+PCR%3A+automatable+amplification+and+sequencing+of+insert+end+fragments+from+P1+and+YAC+clones+for+chromosome+walking&rft.aulast=Y.G.+Liu+and+R.+F.+Whittier&rft.au=Y.G.+Liu+and+R.+F.+Whittier&rft.date=1995&rft.genre=article&rft_id=info%3Adoi%2F10.1016%2F0888-7543%2895%2980010-J&rft_id=info%3Apmid%2F7759102&rft.issue=3&rft.jtitle=Genomics&rft.pages=674-81&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=25"></span></span></li> <li id="cite_note-47"> <span class="reference-text"><span class="citation journal">Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991). <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC328507" rel="nofollow">"<span style="padding-left: 0.2em;">'</span>Touchdown' PCR to circumvent spurious priming during gene amplification"</a>. <i>Nucl Acids Res</i> <b>19</b> (14): 4008. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1093%2Fnar%2F19.14.4008" rel="nofollow">10.1093/nar/19.14.4008</a>. <a href="http://en.wikipedia.org/wiki/PubMed_Central" title="PubMed Central">PMC</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC328507" rel="nofollow">328507</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/1861999" rel="nofollow">1861999</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=%27Touchdown%27+PCR+to+circumvent+spurious+priming+during+gene+amplification&rft.au=Don+RH%2C+Cox+PT%2C+Wainwright+BJ%2C+Baker+K%2C+Mattick+JS&rft.aulast=Don+RH%2C+Cox+PT%2C+Wainwright+BJ%2C+Baker+K%2C+Mattick+JS&rft.date=1991&rft.genre=article&rft_id=%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC328507&rft_id=info%3Adoi%2F10.1093%2Fnar%2F19.14.4008&rft_id=info%3Apmc%2F328507&rft_id=info%3Apmid%2F1861999&rft.issue=14&rft.jtitle=Nucl+Acids+Res&rft.pages=4008&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=19"></span></span></li> <li id="cite_note-Myrick_and_Gelbart-48"> <span class="reference-text"><span class="citation journal">Myrick KV, Gelbart WM (2002). "Universal Fast Walking for direct and versatile determination of flanking sequence". <i>Gene</i> <b>284</b> (1–2): 125–131. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2FS0378-1119%2802%2900384-0" rel="nofollow">10.1016/S0378-1119(02)00384-0</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/11891053" rel="nofollow">11891053</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Universal+Fast+Walking+for+direct+and+versatile+determination+of+flanking+sequence&rft.aulast=Myrick+KV%2C+Gelbart+WM&rft.au=Myrick+KV%2C+Gelbart+WM&rft.date=2002&rft.genre=article&rft_id=info%3Adoi%2F10.1016%2FS0378-1119%2802%2900384-0&rft_id=info%3Apmid%2F11891053&rft.issue=1%E2%80%932&rft.jtitle=Gene&rft.pages=125-131&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=284"></span></span></li> <li id="cite_note-lane_rage-49"> <span class="reference-text"><a class="external text" href="http://www.ejbiotechnology.info/content/vol8/issue2/full/7/index.html" rel="nofollow">Park DJ Electronic Journal of Biotechnology (online). 15 August 2005, vol. 8, no. 2</a></span></li> <li id="cite_note-Parkb-50"> <span class="reference-text"><span class="citation journal">Park DJ (2005). "A new 5' terminal murine GAPDH exon identified using 5'RACE LaNe". <i>Molecular Biotechnology</i> <b>29</b> (1): 39–46. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1385%2FMB%3A29%3A1%3A39" rel="nofollow">10.1385/MB:29:1:39</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/15668518" rel="nofollow">15668518</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=A+new+5%27+terminal+murine+GAPDH+exon+identified+using+5%27RACE+LaNe&rft.aulast=Park+DJ&rft.au=Park+DJ&rft.date=2005&rft.genre=article&rft_id=info%3Adoi%2F10.1385%2FMB%3A29%3A1%3A39&rft_id=info%3Apmid%2F15668518&rft.issue=1&rft.jtitle=Molecular+Biotechnology&rft.pages=39-46&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=29"></span></span></li> <li id="cite_note-Park-51"> <span class="reference-text"><span class="citation journal">Park DJ (2004). "3'RACE LaNe: a simple and rapid fully nested PCR method to determine 3'-terminal cDNA sequence". <i>Biotechniques</i> <b>36</b> (4): 586–588, 590. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/15088375" rel="nofollow">15088375</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=3%27RACE+LaNe%3A+a+simple+and+rapid+fully+nested+PCR+method+to+determine+3%27-terminal+cDNA+sequence&rft.aulast=Park+DJ&rft.au=Park+DJ&rft.date=2004&rft.genre=article&rft_id=info%3Apmid%2F15088375&rft.issue=4&rft.jtitle=Biotechniques&rft.pages=586-588%2C+590&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=36"></span></span></li> <li id="cite_note-52"> <span class="reference-text"><span class="citation journal">Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG (1971). <a class="external text" href="http://www.sciencedirect.com/science/article/pii/0022283671904694" rel="nofollow">"Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases"</a>. <i>J. Mol. Biol.</i> <b>56</b> (2): 341–361. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1016%2F0022-2836%2871%2990469-4" rel="nofollow">10.1016/0022-2836(71)90469-4</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/4927950" rel="nofollow">4927950</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=Studies+on+polynucleotides.+XCVI.+Repair+replications+of+short+synthetic+DNA%27s+as+catalyzed+by+DNA+polymerases&rft.au=Kleppe+K%2C+Ohtsuka+E%2C+Kleppe+R%2C+Molineux+I%2C+Khorana+HG&rft.aulast=Kleppe+K%2C+Ohtsuka+E%2C+Kleppe+R%2C+Molineux+I%2C+Khorana+HG&rft.date=1971&rft.genre=article&rft_id=http%3A%2F%2Fwww.sciencedirect.com%2Fscience%2Farticle%2Fpii%2F0022283671904694&rft_id=info%3Adoi%2F10.1016%2F0022-2836%2871%2990469-4&rft_id=info%3Apmid%2F4927950&rft.issue=2&rft.jtitle=J.+Mol.+Biol.&rft.pages=341-361&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=56"></span></span></li> <li id="cite_note-53"> <span class="reference-text"><span class="citation book"><a href="http://en.wikipedia.org/wiki/Paul_Rabinow" title="Paul Rabinow">Rabinow, Paul</a> (1996). <i>Making PCR: A Story of Biotechnology</i>. Chicago: University of Chicago Press. <a href="http://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="http://en.wikipedia.org/wiki/Special:BookSources/0-226-70146-8" title="Special:BookSources/0-226-70146-8">0-226-70146-8</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.aufirst=Paul&rft.aulast=Rabinow&rft.au=Rabinow%2C+Paul&rft.btitle=Making+PCR%3A+A+Story+of+Biotechnology&rft.date=1996&rft.genre=book&rft.isbn=0-226-70146-8&rft.place=Chicago&rft.pub=University+of+Chicago+Press&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-Mullis-54"> <span class="reference-text"><span class="citation book"><a href="http://en.wikipedia.org/wiki/Kary_Mullis" title="Kary Mullis">Mullis, Kary</a> (1998). <i>Dancing Naked in the Mind Field</i>. New York: Pantheon Books. <a href="http://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="http://en.wikipedia.org/wiki/Special:BookSources/0-679-44255-3" title="Special:BookSources/0-679-44255-3">0-679-44255-3</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.aufirst=Kary&rft.aulast=Mullis&rft.au=Mullis%2C+Kary&rft.btitle=Dancing+Naked+in+the+Mind+Field&rft.date=1998&rft.genre=book&rft.isbn=0-679-44255-3&rft.place=New+York&rft.pub=Pantheon+Books&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook"></span></span></li> <li id="cite_note-55"> <span class="reference-text"><span class="citation journal">Mullis, Kary (1990). "The unusual origin of the polymerase chain reaction". <i>Scientific American</i> <b>262</b> (4): 56–61, 64–5. <a href="http://en.wikipedia.org/wiki/Digital_object_identifier" title="Digital object identifier">doi</a>:<a class="external text" href="http://dx.doi.org/10.1038%2Fscientificamerican0490-56" rel="nofollow">10.1038/scientificamerican0490-56</a>. <a class="mw-redirect" href="http://en.wikipedia.org/wiki/PubMed_Identifier" title="PubMed Identifier">PMID</a> <a class="external text" href="http://www.ncbi.nlm.nih.gov/pubmed/2315679" rel="nofollow">2315679</a>.</span><span class="Z3988" title="ctx_ver=Z39.88-2004&rfr_id=info%3Asid%2Fen.wikipedia.org%3APolymerase+chain+reaction&rft.atitle=The+unusual+origin+of+the+polymerase+chain+reaction&rft.aufirst=Kary&rft.aulast=Mullis&rft.au=Mullis%2C+Kary&rft.date=1990&rft.genre=article&rft_id=info%3Adoi%2F10.1038%2Fscientificamerican0490-56&rft_id=info%3Apmid%2F2315679&rft.issue=4&rft.jtitle=Scientific+American&rft.pages=56-61%2C+64-5&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.volume=262"></span></span></li> <li id="cite_note-56"> <span class="reference-text"><a class="external text" href="http://www.genengnews.com/articles/chitem.aspx?aid=1656&chid=0" rel="nofollow">Advice on How to Survive the Taq Wars ¶2</a>: <a class="new" href="http://en.wikipedia.org/w/index.php?title=GEN_Genetic_Engineering_News&action=edit&redlink=1" title="GEN Genetic Engineering News (page does not exist)">GEN Genetic Engineering News</a> Biobusiness Channel: Article. May 1, 2006 (Vol. 26, No. 9).</span></li> </ol> </div> <h2> <span class="mw-headline" id="External_links">External links</span></h2> <table cellpadding="0" cellspacing="5" class="vertical-navbox nowraplinks infobox" style="background: #f9f9f9; border-spacing: 0.4em 0; border: 1px solid #aaa; clear: right; float: right; font-size: 88%; line-height: 1.4em; margin: 0 0 1.0em 1.0em; padding: 0.2em; text-align: center; width: auto;"> <tbody> <tr> <td style="line-height: 1.2em; padding-top: 0.4em;"><a class="mw-redirect" href="http://en.wikipedia.org/wiki/Wikipedia:LIBRARY" title="Wikipedia:LIBRARY">Library resources</a> about<br /> <b>Polymerase chain reaction</b> <hr /> </td> </tr> <tr> <td class="plainlist" style="padding: 0 0.1em 0.4em; text-align: left;"> <ul> <li><a class="external text" href="http://tools.wmflabs.org/ftl/cgi-bin/ftl?st=&su=Polymerase+chain+reaction">Resources in your library</a></li> <li><a class="external text" href="http://tools.wmflabs.org/ftl/cgi-bin/ftl?st=&su=Polymerase+chain+reaction&library=0CHOOSE0">Resources in other libraries</a></li> </ul> </td> </tr> </tbody></table> <table class="metadata mbox-small plainlinks" style="background-color: #f9f9f9; border: 1px solid #aaa;"> <tbody> <tr> <td class="mbox-image"><img alt="" data-file-height="1376" data-file-width="1024" height="40" src="http://upload.wikimedia.org/wikipedia/en/thumb/4/4a/Commons-logo.svg/30px-Commons-logo.svg.png" srcset="//upload.wikimedia.org/wikipedia/en/thumb/4/4a/Commons-logo.svg/45px-Commons-logo.svg.png 1.5x, //upload.wikimedia.org/wikipedia/en/thumb/4/4a/Commons-logo.svg/59px-Commons-logo.svg.png 2x" width="30" /></td> <td class="mbox-text plainlist">Wikimedia Commons has media related to <i><b><a class="extiw" href="http://commons.wikimedia.org/wiki/Polymerase_chain_reaction" title="commons:Polymerase chain reaction">Polymerase chain reaction</a></b></i>.</td> </tr> </tbody></table> <ul> <li><a class="external text" href="http://www.selectscience.net/pcr_buying_guide.aspx" rel="nofollow">A Guide to PCR Technologies</a> SelectScience</li> <li><a class="external text" href="http://openpcr.org/" rel="nofollow">OpenPCR</a> Open-source PCR thermalcycler project</li> <li><a class="external text" href="http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&d=PALL&RefSrch=yes&Query=PN%2F4683202" rel="nofollow">US Patent for PCR</a></li> <li><a class="external text" href="http://www.dnalc.org/ddnalc/resources/pcr.html" rel="nofollow">Step-through animation of PCR</a> - <a href="http://en.wikipedia.org/wiki/Cold_Spring_Harbor_Laboratory" title="Cold Spring Harbor Laboratory">Cold Spring Harbor Laboratory</a></li> <li><a class="external text" href="http://openwetware.org/wiki/PCR" rel="nofollow">OpenWetWare</a></li> <li><a class="external text" href="https://www.youtube.com/watch?v=rbvvvLWE8jI" rel="nofollow">What is PCR plateau effect?</a> YouTube tutorial video</li> <li><a class="external text" href="http://siarchives.si.edu/collections/siris_arc_217745" rel="nofollow">History of the Polymerase Chain Reaction</a> from the <a href="http://en.wikipedia.org/wiki/Smithsonian_Institution_Archives" title="Smithsonian Institution Archives">Smithsonian Institution Archives</a></li> <li><a class="external text" href="http://www.thingiverse.com/search?q=pcr&sa=" rel="nofollow">3d models of PCR equipment for 3D printing on thingiverse.com</a></li> <li><a class="external text" href="http://insilico.ehu.es/edu/PCR_en/" rel="nofollow">Computer exercise. Design of PCR and PCR-RFLP experiments</a></li> </ul> <div> From Wikipedia--------------------------------------------------------</div> </div> </div> </div> </div>

What is PCR ? Polymerase chain reaction

Polymerase chain reaction "PCR" redirects here. For other uses, see PCR (disambiguati...

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