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Data management in clinical research: An overview - Biotech Freshers must learn or Call us for any query "D10"

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Abstract</h2>
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Clinical Data Management (CDM) is a critical phase in clinical research, which leads to generation of high-quality, reliable, and statistically sound data from clinical trials. This helps to produce a drastic reduction in time from drug development to marketing. Team members of CDM are actively involved in all stages of clinical trial right from inception to completion. They should have adequate process knowledge that helps maintain the quality standards of CDM processes. Various procedures in CDM including Case Report Form (CRF) designing, CRF annotation, database designing, data-entry, data validation, discrepancy management, medical coding, data extraction, and database locking are assessed for quality at regular intervals during a trial. In the present scenario, there is an increased demand to improve the CDM standards to meet the regulatory requirements and stay ahead of the competition by means of faster commercialization of product. With the implementation of regulatory compliant data management tools, CDM team can meet these demands. Additionally, it is becoming mandatory for companies to submit the data electronically. CDM professionals should meet appropriate expectations and set standards for data quality and also have a drive to adapt to the rapidly changing technology. This article highlights the processes involved and provides the reader an overview of the tools and standards adopted as well as the roles and responsibilities in CDM.</div>
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<strong class="kwd-title">KEY WORDS: </strong><span class="kwd-text">Clinical data interchange standards consortium, clinical data management systems, data management, e-CRF, good clinical data management practices, validation</span></div>
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Introduction</h2>
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Clinical trial is intended to find answers to the research question by means of generating data for proving or disproving a hypothesis. The quality of data generated plays an important role in the outcome of the study. Often research students ask the question, “what is Clinical Data Management (CDM) and what is its significance?” Clinical data management is a relevant and important part of a clinical trial. All researchers try their hands on CDM activities during their research work, knowingly or unknowingly. Without identifying the technical phases, we undertake some of the processes involved in CDM during our research work. This article highlights the processes involved in CDM and gives the reader an overview of how data is managed in clinical trials.</div>
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CDM is the process of collection, cleaning, and management of subject data in compliance with regulatory standards. The primary objective of CDM processes is to provide high-quality data by keeping the number of errors and missing data as low as possible and gather maximum data for analysis.[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode tag_hotlink tag_tooltip jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref1" id="__tag_257656277" rid="ref1" role="button">1</a>] To meet this objective, best practices are adopted to ensure that data are complete, reliable, and processed correctly. This has been facilitated by the use of software applications that maintain an audit trail and provide easy identification and resolution of data discrepancies. Sophisticated innovations[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref2" rid="ref2" role="button">2</a>] have enabled CDM to handle large trials and ensure the data quality even in complex trials.</div>
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How do we define ‘high-quality’ data? High-quality data should be absolutely accurate and suitable for statistical analysis. These should meet the protocol-specified parameters and comply with the protocol requirements. This implies that in case of a deviation, not meeting the protocol-specifications, we may think of excluding the patient from the final database. It should be borne in mind that in some situations, regulatory authorities may be interested in looking at such data. Similarly, missing data is also a matter of concern for clinical researchers. High-quality data should have minimal or no misses. But most importantly, high-quality data should possess only an arbitrarily ‘acceptable level of variation’ that would not affect the conclusion of the study on statistical analysis. The data should also meet the applicable regulatory requirements specified for data quality.</div>
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Tools for CDM</h4>
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Many software tools are available for data management, and these are called Clinical Data Management Systems (CDMS). In multicentric trials, a CDMS has become essential to handle the huge amount of data. Most of the CDMS used in pharmaceutical companies are commercial, but a few open source tools are available as well. Commonly used CDM tools are ORACLE CLINICAL, CLINTRIAL, MACRO, RAVE, and eClinical Suite. In terms of functionality, these software tools are more or less similar and there is no significant advantage of one system over the other. These software tools are expensive and need sophisticated Information Technology infrastructure to function. Additionally, some multinational pharmaceutical giants use custom-made CDMS tools to suit their operational needs and procedures. Among the open source tools, the most prominent ones are OpenClinica, openCDMS, TrialDB, and PhOSCo. These CDM software are available free of cost and are as good as their commercial counterparts in terms of functionality. These open source software can be downloaded from their respective websites.</div>
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In regulatory submission studies, maintaining an audit trail of data management activities is of paramount importance. These CDM tools ensure the audit trail and help in the management of discrepancies. According  to the roles and responsibilities (explained later), multiple user IDs can be created with access limitation to data entry, medical coding, database designing, or quality check. This ensures that each user can access only the respective functionalities allotted to that user ID and cannot make any other change in the database. For responsibilities where changes are permitted to be made in the data, the software will record the change made, the user ID that made the change and the time and date of change, for audit purposes (audit trail). During a regulatory audit, the auditors can verify the discrepancy management process; the changes made and can confirm that no unauthorized or false changes were made.</div>
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Regulations, Guidelines, and Standards in CDM</h4>
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Akin to other areas in clinical research, CDM has guidelines and standards that must be followed. Since the pharmaceutical industry relies on the electronically captured data for the evaluation of medicines, there is a need to follow good practices in CDM and maintain standards in electronic data capture. These electronic records have to comply with a Code of Federal Regulations (CFR), 21 CFR Part 11. This regulation is applicable to records in electronic format that are created, modified, maintained, archived, retrieved, or transmitted. This demands the use of validated systems to ensure accuracy, reliability, and consistency of data with the use of secure, computer-generated, time-stamped audit trails to independently record the date and time of operator entries and actions that create, modify, or delete electronic records.[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref3" rid="ref3" role="button">3</a>] Adequate procedures and controls should be put in place to ensure the integrity, authenticity, and confidentiality of data. If data have to be submitted to regulatory authorities, it should be entered and processed in 21 CFR part 11-compliant systems. Most of the CDM systems available are like this and pharmaceutical companies as well as contract research organizations ensure this compliance.</div>
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Society for Clinical Data Management (SCDM) publishes the Good Clinical Data Management Practices (GCDMP) guidelines, a document providing the standards of good practice within CDM. GCDMP was initially published in September 2000 and has undergone several revisions thereafter. The July 2009 version is the currently followed GCDMP document. GCDMP provides guidance on the accepted practices in CDM that are consistent with regulatory practices. Addressed in 20 chapters, it covers the CDM process by highlighting the minimum standards and best practices.</div>
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Clinical Data Interchange Standards Consortium (CDISC), a multidisciplinary non-profit organization, has developed standards to support acquisition, exchange, submission, and archival of clinical research data and metadata. Metadata is the data of the data entered. This includes data about the individual who made the entry or a change in the clinical data, the date and time of entry/change and details of the changes that have been made. Among the standards, two important ones are the Study Data Tabulation Model Implementation Guide for Human Clinical Trials (SDTMIG) and the Clinical Data Acquisition Standards Harmonization (CDASH) standards, available free of cost from the CDISC website (<a href="http://www.cdisc.org/" ref="reftype=extlink&article-id=3326906&issue-id=208917&journal-id=979&FROM=Article%7CBody&TO=External%7CLink%7CURI&rendering-type=normal" target="pmc_ext">www.cdisc.org</a>). The SDTMIG standard[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref4" rid="ref4" role="button">4</a>] describes the details of model and standard terminologies for the data and serves as a guide to the organization. CDASH v 1.1[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref5" rid="ref5" role="button">5</a>] defines the basic standards for the collection of data in a clinical trial and enlists the basic data information needed from a clinical, regulatory, and scientific perspective.</div>
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The CDM Process</h4>
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The CDM process, like a clinical trial, begins with the end in mind. This means that the whole process is designed keeping the deliverable in view. As a clinical trial is designed to answer the research question, the CDM process is designed to deliver an error-free, valid, and statistically sound database. To meet this objective, the CDM process starts early, even before the finalization of the study protocol.</div>
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Review and finalization of study documents</h4>
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The protocol is reviewed from a database designing perspective, for clarity and consistency. During this review, the CDM personnel will identify the data items to be collected and the frequency of collection with respect to the visit schedule. A Case Report Form (CRF) is designed by the CDM team, as this is the first step in translating the protocol-specific activities into data being generated. The data fields should be clearly defined and be consistent throughout. The type of data to be entered should be evident from the CRF. For example, if weight has to be captured in two decimal places, the data entry field should have two data boxes placed after the decimal as shown in <a class="fig-table-link fig figpopup" co-legend-rid="lgnd_F1" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/figure/F1/" rid-figpopup="F1" rid-ob="ob-F1" target="figure">Figure 1</a>. Similarly, the units in which measurements have to be made should also be mentioned next to the data field. The CRF should be concise, self-explanatory, and user-friendly (unless you are the one entering data into the CRF). Along with the CRF, the filling instructions (called CRF Completion Guidelines) should also be provided to study investigators for error-free data acquisition. CRF annotation is done wherein the variable is named according to the SDTMIG or the conventions followed internally. Annotations are coded terms used in CDM tools to indicate the variables in the study. An example of an annotated CRF is provided in <a class="fig-table-link fig figpopup" co-legend-rid="lgnd_F1" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/figure/F1/" rid-figpopup="F1" rid-ob="ob-F1" target="figure">Figure 1</a>. In questions with discrete value options (like the variable gender having values male and female as responses), all possible options will be coded appropriately.</div>
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<a class="figpopup" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/figure/F1/" rid-figpopup="F1" rid-ob="ob-F1" target="figure">Figure 1</a></div>
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Annotated sample of a Case Report Form (CRF). Annotations are entered in coloured text in this figure to differentiate from the CRF questions. DCM = Data collection module, DVG = Discrete value group, YNNA [S1] = Yes, No = Not applicable [subset 1], C <strong>...</strong></div>
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Based on these, a Data Management Plan (DMP) is developed. DMP document is a road map to handle the data under foreseeable circumstances and describes the CDM activities to be followed in the trial. A list of CDM activities is provided in <a class="fig-table-link table figpopup" co-legend-rid="" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/table/T1/" rid-figpopup="T1" rid-ob="ob-T1" target="true">Table 1</a>. The DMP describes the database design, data entry and data tracking guidelines, quality control measures, SAE reconciliation guidelines, discrepancy management, data transfer/extraction, and database locking guidelines. Along with the DMP, a Data Validation Plan (DVP) containing all edit-checks to be performed and the calculations for derived variables are also prepared. The edit check programs in the DVP help in cleaning up the data by identifying the discrepancies.</div>
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<a class="table img_link icnblk_img figpopup" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/table/T1/" rid-figpopup="T1" rid-ob="ob-T1" target="table"><img alt="Table 1" class="small-thumb" src="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/table/T1/?report=thumb" src-large="/pmc/articles/PMC3326906/table/T1/?report=previmg" title="Table 1" /></a><div class="icnblk_cntnt">
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<a class="figpopup" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/table/T1/" rid-figpopup="T1" rid-ob="ob-T1" target="table">Table 1</a></div>
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List of clinical data management activities</div>
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Database designing</h4>
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Databases are the clinical software applications, which are built to facilitate the CDM tasks to carry out multiple studies.[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode tag_hotlink tag_tooltip jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref6" id="__tag_257656273" rid="ref6" role="button">6</a>] Generally, these tools have built-in compliance with regulatory requirements and are easy to use. “System validation” is conducted to ensure data security, during which system specifications,[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode tag_hotlink tag_tooltip jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref7" id="__tag_257656272" rid="ref7" role="button">7</a>] user requirements, and regulatory compliance are evaluated before implementation. Study details like objectives, intervals, visits, investigators, sites, and patients are defined in the database and CRF layouts are designed for data entry. These entry screens are tested with dummy data before moving them to the real data capture.</div>
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Data collection</h4>
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Data collection is done using the CRF that may exist in the form of a paper or an electronic version. The traditional method is to employ paper CRFs to collect the data responses, which are translated to the database by means of data entry done in-house. These paper CRFs are filled up by the investigator according to the completion guidelines. In the e-CRF-based CDM, the investigator or a designee will be logging into the CDM system and entering the data directly at the site. In e-CRF method, chances of errors are less, and the resolution of discrepancies happens faster. Since pharmaceutical companies try to reduce the time taken for drug development processes by enhancing the speed of processes involved, many pharmaceutical companies are opting for e-CRF options (also called remote data entry).</div>
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CRF tracking</h4>
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The entries made in the CRF will be monitored by the Clinical Research Associate (CRA) for completeness and filled up CRFs are retrieved and handed over to the CDM team. The CDM team will track the retrieved CRFs and maintain their record. CRFs are tracked for missing pages and illegible data manually to assure that the data are not lost. In case of missing or illegible data, a clarification is obtained from the investigator and the issue is resolved.</div>
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Data entry</h4>
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Data entry takes place according to the guidelines prepared along with the DMP. This is applicable only in the case of paper CRF retrieved from the sites. Usually, double data entry is performed wherein the data is entered by two operators separately.[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode tag_hotlink tag_tooltip jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref8" id="__tag_257656276" rid="ref8" role="button">8</a>] The second pass entry (entry made by the second person) helps in verification and reconciliation by identifying the transcription errors and discrepancies caused by illegible data. Moreover, double data entry helps in getting a cleaner database compared to a single data entry. Earlier studies have shown that double data entry ensures better consistency with paper CRF as denoted by a lesser error rate.[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode tag_hotlink tag_tooltip jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref9" id="__tag_257656274" rid="ref9" role="button">9</a>]</div>
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Data validation</h4>
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Data validation is the process of testing the validity of data in accordance with the protocol specifications. Edit check programs are written to identify the discrepancies in the entered data, which are embedded in the database, to ensure data validity. These programs are written according to the logic condition mentioned in the DVP. These edit check programs are initially tested with dummy data containing discrepancies. Discrepancy is defined as a data point that fails to pass a validation check. Discrepancy may be due to inconsistent data, missing data, range checks, and deviations from the protocol. In e-CRF based studies, data validation process will be run frequently for identifying discrepancies. These discrepancies will be resolved by investigators after logging into the system. Ongoing quality control of data processing is undertaken at regular intervals during the course of CDM. For example, if the inclusion criteria specify that the age of the patient should be between 18 and 65 years (both inclusive), an edit program will be written for two conditions <em>viz</em>. age <18 and >65. If for any patient, the condition becomes TRUE, a discrepancy will be  generated. These discrepancies will be highlighted in the system and Data Clarification Forms (DCFs) can be generated. DCFs are documents containing queries pertaining to the discrepancies identified.</div>
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Discrepancy management</h4>
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This is also called query resolution. Discrepancy management includes reviewing discrepancies, investigating the reason, and resolving them with documentary proof or declaring them as irresolvable. Discrepancy management helps in cleaning the data and gathers enough evidence for the deviations observed in data. Almost all CDMS have a discrepancy database where all discrepancies will be recorded and stored with audit trail.</div>
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Based on the types identified, discrepancies are either flagged to the investigator for clarification or closed in-house by Self-Evident Corrections (SEC) without sending DCF to the site. The most common SECs are obvious spelling errors. For discrepancies that require clarifications from the investigator, DCFs will be sent to the site. The CDM tools help in the creation and printing of DCFs. Investigators will write the resolution or explain the circumstances that led to the discrepancy in data. When a resolution is provided by the investigator, the same will be updated in the database. In case of e-CRFs, the investigator can access the discrepancies flagged to him and will be able to provide the resolutions online. <a class="fig-table-link fig figpopup" co-legend-rid="lgnd_F2" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/figure/F2/" rid-figpopup="F2" rid-ob="ob-F2" target="figure">Figure 2</a> illustrates the flow of discrepancy management.</div>
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Discrepancy management (DCF = Data clarification form, CRA = Clinical Research Associate, SDV = Source document verification, SEC = Self-evident correction)</div>
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The CDM team reviews all discrepancies at regular intervals to ensure that they have been resolved. The resolved data discrepancies are recorded as ‘closed’. This means that those validation failures are no longer considered to be active, and future data validation attempts on the same data will not create a discrepancy for same data point. But closure of discrepancies is not always possible. In some cases, the investigator will not be able to provide a resolution for the discrepancy. Such discrepancies will be considered as ‘irresolvable’ and will be updated in the discrepancy database.</div>
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Discrepancy management is the most critical activity in the CDM process. Being the vital activity in cleaning up the data, utmost attention must be observed while handling the discrepancies.</div>
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Medical coding</h4>
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Medical coding helps in identifying and properly classifying the medical terminologies associated with the clinical trial. For classification of events, medical dictionaries available online are used. Technically, this activity needs the knowledge of medical terminology, understanding of disease entities, drugs used, and a basic knowledge of the pathological processes involved. Functionally, it also requires knowledge about the structure of electronic medical dictionaries and the hierarchy of classifications available in them. Adverse events occurring during the study, prior to and concomitantly administered medications and pre-or co-existing illnesses are coded using the available medical dictionaries. Commonly, Medical Dictionary for Regulatory Activities (MedDRA) is used for the coding of adverse events as well as other illnesses and World Health Organization–Drug Dictionary Enhanced (WHO-DDE) is used for coding the medications. These dictionaries contain the respective classifications of adverse events and drugs in proper classes. Other dictionaries are also available for use in data management (eg, WHO-ART is a dictionary that deals with adverse reactions terminology). Some pharmaceutical companies utilize customized dictionaries to suit their needs and meet their standard operating procedures.</div>
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Medical coding helps in classifying reported medical terms on the CRF to standard dictionary terms in order to achieve data consistency and avoid unnecessary duplication. For example, the investigators may use different terms for the same adverse event, but it is important to code all of them to a single standard code and maintain uniformity in the process. The right coding and classification of adverse events and medication is crucial as an incorrect coding may lead to masking of safety issues or highlight the wrong safety concerns related to the drug.</div>
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Database locking</h4>
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After a proper quality check and assurance, the final data validation is run. If there are no discrepancies, the SAS datasets are finalized in consultation with the statistician. All data management activities should have been completed prior to database lock. To ensure this, a pre-lock checklist is used and completion of all activities is confirmed. This is done as the database cannot be changed in any manner after locking. Once the approval for locking is obtained from all stakeholders, the database is locked and clean data is extracted for statistical analysis. Generally, no modification in the database is possible. But in case of a critical issue or for other important operational reasons, privileged users can modify the data even after the database is locked. This, however, requires proper documentation and an audit trail has to be maintained with sufficient justification for updating the locked database. Data extraction is done from the final database after locking. This is followed by its archival.</div>
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Roles and Responsibilities in CDM</h4>
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In a CDM team, different roles and responsibilities are attributed to the team members. The minimum educational requirement for a team member in CDM should be graduation in life science and knowledge of computer applications. Ideally, medical coders should be medical graduates. However, in the industry, paramedical graduates are also recruited as medical coders. Some key roles are essential to all CDM teams. The list of roles given below can be considered as minimum requirements for a CDM team:</div>
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<li>Data Manager</li>
<li>Database Programmer/Designer</li>
<li>Medical Coder</li>
<li>Clinical Data Coordinator</li>
<li>Quality Control Associate</li>
<li>Data Entry Associate</li>
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The data manager is responsible for supervising the entire CDM process. The data manager prepares the DMP, approves the CDM procedures and all internal documents related to CDM activities. Controlling and allocating the database access to team members is also the responsibility of the data manager. The database programmer/designer performs the CRF annotation, creates the study database, and programs the edit checks for data validation. He/she is also responsible for designing of data entry screens in the database and validating the edit checks with dummy data. The medical coder will do the coding for adverse events, medical history, co-illnesses, and concomitant medication administered during the study. The clinical data coordinator designs the CRF, prepares the CRF filling instructions, and is responsible for developing the DVP and discrepancy management. All other CDM-related documents, checklists, and guideline documents are prepared by the clinical data coordinator. The quality control associate checks the accuracy of data entry and conducts data audits.[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode tag_hotlink tag_tooltip jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref10" id="__tag_257656271" rid="ref10" role="button">10</a>] Sometimes, there is a separate quality assurance person to conduct the audit on the data entered. Additionally, the quality control associate verifies the documentation pertaining to the procedures being followed. The data entry personnel will be tracking the receipt of CRF pages and performs the data entry into the database.</div>
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Conclusion</h2>
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CDM has evolved in response to the ever-increasing demand from pharmaceutical companies to fast-track the drug development process and from the regulatory authorities to put the quality systems in place to ensure generation of high-quality data for accurate drug evaluation. To meet the expectations, there is a gradual shift from the paper-based to the electronic systems of data management. Developments on the technological front have positively impacted the CDM process and systems, thereby leading to encouraging results on speed and quality of data being generated. At the same time, CDM professionals should ensure the standards for improving data quality.[<a aria-expanded="false" aria-haspopup="true" class="bibr popnode tag_hotlink tag_tooltip jig-ncbipopper" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326906/#ref11" id="__tag_257656275" rid="ref11" role="button">11</a>] CDM, being a speciality in itself, should be evaluated by means of the systems and processes being implemented and the standards being followed. The biggest challenge from the regulatory perspective would be the standardization of data management process across organizations, and development of regulations to define the procedures to be followed and the data standards. From the industry perspective, the biggest hurdle would be the planning and implementation of data management systems in a changing operational environment where the rapid pace of technology development outdates the existing infrastructure. In spite of these, CDM is evolving to become a standard-based clinical research entity, by striking a balance between the expectations from and constraints in the existing systems, driven by technological developments and business demands.</div>
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Abstract Clinical Data Management (CDM) is a critical phase in clinical research, which leads to generation of high-quality, reliable,...

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Bio-Technology is a research oriented science, a combination of Biology and Technology. It covers a wide variety of subjects like Genetics, Biochemistry, Microbiology, Immunology, Virology, Chemistry and Engineering and is also concerned with many other subjects like Health and  Medicine, Agriculture and Animal Husbandry, Cropping system and Crop Management, Ecology, Cell Biology, Soil science<br />
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Job Description  CRA 1-1411760 Description  The CRA conducts monitoring visits for assigned protocols and study sites. Overall responsibilities are to ensure that the study is being conducted in accordance with the protocol, standard operating procedures, good clinical practice, and applicable regulatory requirements. - Performs site selection, initiation, monitoring and close<br />
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Clinical Data Coordinator I @ ICON || HD10<br />
7/18/2014<br />
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Description DEPARTMENT: Data Management JOB TITLE: Clinical Data Coordinator I REPORTS TO: Data Management Project Lead, Data Management Project Manager SECTION 1: POSITION SUMMARY Under supervision of more experienced Data Management Personnel and<br />
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Defence Institute of Physiology and Allied Sciences (DIPAS) invites applications for Junior Research Fellows and Research Associate Defence Institute of Physiology and Allied Sciences (DIPAS) provides an excellent opportunity for young and motivated researchers to pursue a career in basic and applied Life Sciences research. DIPAS endeavors in optimizing human performance under<br />
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7/18/2014<br />
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Eligible candidates are invited to attend WALK - IN - INTERVIEW on the following date at 9.30 a.m. sharp for the following positions on the ad hoc research project entitled “Vitamin A metabolic pathway on region – specific adipose tissue development and its relevance to insulin resistance: A proteomic approach to sexual dimorphism”. The essential qualification, experience, consolidated<br />
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Research Associate and Junior Research Fellow in Bharathidasan University || HD5<br />
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Bharathidasan University established in February 1982, and was named after the great revolutionary Tamil Poet, Bharathidasan (1891-1968). The motto of the University "We will create a brave new world". The University endeavours to be true to such a vision by creating in the region a brave new world of academic innovation for social change"(NAAC, - 2005, p.69). The year 2006-07 is the<br />
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Opening for Medical Representative in Soranus Lifesciences | Palanpur || HD4<br />
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ZONAL SALES MANAGER {Ophthgalmic div} in Reputed Pharma client of Nextus Solutions || HD3<br />
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One of the reputed pharma group of India with presence in more than 30 countries globally. Reputed client of Nextus Solutions. Nextus Solutions is one of the leading training and placement provider in Pharma industry. Post: ZONAL SALES MANAGER {Ophthgalmic div} Job Description Has to handle South and WEST Zone. Its equivalent to third line manager Should have experience of ophthalmic<br />
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Regional Plant Resource Centre requires Junior Research Fellow, Senior Research Fellow Regional Plant Resource Centre was established in 1985 as a recreational garden and with an aim to provide a green lung for the inhabitants of Bhubaneswar – the capital city of Orissa. Gradually it became a dynamic and vibrant research organization of the State<br />
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Career for M.Pharm/M.Tech to work in project under DBTs NER TWINNING Programme in collaboration with NIMHANS and ICGEB || HD1<br />
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Assam University came into existence through Assam (Central) University Act, 1989. It was established in 1994, since then the University is steadily becoming an excellent centre of learning. The campus, situated in Dargakona, about 20 kms from  Silchar, is set amid sprawling hillocks, a typical landscape in the  North East. The campus  in an area of  600 acres surrounded by lakes and<br />
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7/18/2014<br />
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Vacancy For: Manager SCM (procurement of Direct Inputs for Production) at Dr. Reddy, Hydrabad B8<br />
7/17/2014<br />
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Medical Representative @ DRL, PAN India B7<br />
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What is PCR ? Polymerase chain reaction

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<span dir="auto">Polymerase chain reaction</span></h1>
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"PCR" redirects here. For other uses, see <a href="http://en.wikipedia.org/wiki/PCR_%28disambiguation%29" title="PCR (disambiguation)">PCR (disambiguation)</a>.</div>
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<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>
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A strip of eight PCR tubes, each containing a 100 μl reaction mixture</div>
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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 />

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Placing a strip of eight PCR tubes, each containing a 100 μl reaction mixture, into the thermal cycler</div>
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Contents</h2>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<h2>
<span class="mw-headline" id="PCR_principles_and_procedure">PCR principles and procedure</span></h2>
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<b>Figure 1a</b>: A thermal cycler for PCR</div>
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<b>Figure 1b</b>: An older model three-temperature thermal cycler for PCR</div>
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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 Mg<sup>2+</sup> is used, but Mn<sup>2+</sup> 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 Mn<sup>2+</sup> 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 />

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<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>
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<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 />

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<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>
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<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<sub>m</sub> of the primers used, while at the later cycles, it is a few degrees (3-5 °C) below the primer T<sub>m</sub>.
 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>
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<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>
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<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 />

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<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>
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"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,
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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,
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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>
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<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>
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<h2>
<span class="mw-headline" id="External_links">External links</span></h2>
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<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>
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</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>
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</tr>
</tbody></table>
<table class="metadata mbox-small plainlinks" style="background-color: #f9f9f9; border: 1px solid #aaa;">
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<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>
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What is PCR ? Polymerase chain reaction

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