Editing Polymerase chain reaction (section)

===Amplification and quantification of DNA===
{{See also|Use of DNA in forensic entomology}}

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 [[forensic analysis]], when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of [[ancient DNA]] 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 [[mammoth]], and also on human DNA, in applications ranging from the analysis of Egyptian [[mummy|mummies]] to the identification of a Russian [[tsar]] and the body of English king [[Richard III]].<ref>{{cite web| url= http://photoscience.la.asu.edu/photosyn/courses/BIO_343/lecture/DNAtech.html| archive-url=https://web.archive.org/web/19971009144333/http://photoscience.la.asu.edu/photosyn/courses/BIO_343/lecture/DNAtech.html| url-status=dead| archive-date=9 October 1997| title= Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343)| publisher=Arizona State University| access-date=2007-10-29}}</ref>

[[Quantitative PCR]] or Real Time PCR (qPCR,<ref>{{cite journal | vauthors = Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT | title = The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments | journal = Clinical Chemistry | volume = 55 | issue = 4 | pages = 611–22 | date = April 2009 | pmid = 19246619 | doi = 10.1373/clinchem.2008.112797 | url = http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf | doi-access = free }}</ref> not to be confused with [[Reverse transcription polymerase chain reaction|RT-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 [[gene expression]]. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method consists of using [[fluorophore|fluorescent]] dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its [[complementary DNA]] (cDNA) takes place. An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique, called RT-qPCR, allows for the quantification of a small quantity of RNA. Through this combined technique, mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of PCR,<ref name="Garibyan, Avashia 1–4">{{cite journal | vauthors = Garibyan L, Avashia N | title = Polymerase chain reaction | journal = The Journal of Investigative Dermatology | volume = 133 | issue = 3 | pages = 1–4 | date = March 2013 | pmid = 23399825 | pmc = 4102308 | doi = 10.1038/jid.2013.1 }}</ref> increasing chances for detection of genes associated with genetic diseases such as cancer.<ref name="Ninfa-2009"/> Laboratories use RT-qPCR for the purpose of sensitively measuring gene regulation. The mathematical foundations for the reliable quantification of the PCR<ref>{{Cite journal|last1=Schnell|first1=S.|last2=Mendoza|first2=C.|date=October 1997|title=Theoretical Description of the Polymerase Chain Reaction|url=https://linkinghub.elsevier.com/retrieve/pii/S0022519397904732|journal=Journal of Theoretical Biology|language=en|volume=188|issue=3|pages=313–18|doi=10.1006/jtbi.1997.0473|pmid=9344735|bibcode=1997JThBi.188..313S|url-access=subscription}}</ref> and RT-qPCR<ref>{{Cite journal|last1=Schnell|first1=S.|last2=Mendoza|first2=C.|date=1997-02-21|title=Enzymological Considerations for the Theoretical Description of the Quantitative Competitive Polymerase Chain Reaction (QC-PCR)|url=http://www.sciencedirect.com/science/article/pii/S0022519396902830|journal=Journal of Theoretical Biology|language=en|volume=184|issue=4|pages=433–40|doi=10.1006/jtbi.1996.0283|pmid=9082073|bibcode=1997JThBi.184..433S|issn=0022-5193|url-access=subscription}}</ref> facilitate the implementation of accurate fitting procedures of experimental data in research, medical, diagnostic and infectious disease applications.<ref>{{Cite journal|last1=Becker|first1=Sven|last2=Böger|first2=Peter|last3=Oehlmann|first3=Ralfh|last4=Ernst|first4=Anneliese|date=2000-11-01|title=PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities|journal=Applied and Environmental Microbiology|language=en|volume=66|issue=11|pages=4945–53|doi=10.1128/AEM.66.11.4945-4953.2000|issn=1098-5336|pmc=92404|pmid=11055948|bibcode=2000ApEnM..66.4945B}}</ref><ref>{{Cite journal|last1=Solomon|first1=Anthony W.|last2=Peeling|first2=Rosanna W.|last3=Foster|first3=Allen|last4=Mabey|first4=David C. W.|date=2004-10-01|title=Diagnosis and Assessment of Trachoma|journal=Clinical Microbiology Reviews|language=en|volume=17|issue=4|pages=982–1011|doi=10.1128/CMR.17.4.982-1011.2004|issn=0893-8512|pmc=523557|pmid=15489358}}</ref><ref>{{Cite journal|last=Ramzy|first=Reda M.R.|date=April 2002|title=Recent advances in molecular diagnostic techniques for human lymphatic filariasis and their use in epidemiological research|url=https://academic.oup.com/trstmh/article-lookup/doi/10.1016/S0035-9203(02)90080-5|journal=Transactions of the Royal Society of Tropical Medicine and Hygiene|language=en|volume=96|pages=S225–29|doi=10.1016/S0035-9203(02)90080-5|pmid=12055843|url-access=subscription}}</ref><ref>{{cite book |last=Sachse |first=Konrad |title=PCR Detection of Microbial Pathogens |chapter=Specificity and Performance of Diagnostic PCR Assays |date=2003 |pages=3–29 |editor-last=Sachse |editor-first=Konrad |series=Methods in Molecular Biology |volume=216 |place=Totowa, New Jersey |publisher=Humana Press |doi=10.1385/1-59259-344-5:03 |pmid=12512353 |isbn=978-1-59259-344-6 |editor2-last=Frey |editor2-first=Joachim }}</ref>