Editing Polymerase chain reaction (section)

==Applications==

===Selective DNA isolation===
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many ways, such as generating [[hybridization probe]]s for [[Southern blot|Southern]] or [[Northern blot|northern]] hybridization and [[DNA cloning]], 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.{{citation needed|date=August 2024}}

Other applications of PCR include [[DNA sequencing]] 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 recombinant DNA technologies involving the insertion of a DNA sequence into a [[plasmid]], [[phage]], or [[cosmid]] (depending on size) or the genetic material of another organism. Bacterial colonies ''(such as [[Escherichia coli|E. coli]])'' can be rapidly screened by PCR for correct DNA [[plasmid|vector]] constructs.<ref name="Hybrid">{{cite book |vauthors=Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI |year= 2006|chapter=Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes|title=DNA Sequencing II: Optimizing Preparation and Cleanup|editor=Kieleczawa J|publisher= Jones & Bartlett|pages=241–57|isbn= 978-0-7637-3383-4}}</ref> PCR may also be used for [[genetic fingerprinting]]; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.{{citation needed|date=August 2024}}

[[File:Pcr fingerprint.png|thumb|upright|Electrophoresis of PCR-amplified DNA fragments: {{Ordered list|Father|Child|Mother}}<br />The child has inherited some, but not all, of the fingerprints of each of its parents, giving it a new, unique fingerprint.]]

Some PCR fingerprint 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 when certain [[molecular clock]]s are used (i.e. the [[16S rRNA]] and recA genes of microorganisms).<ref>{{cite journal | vauthors = Pombert JF, Sistek V, Boissinot M, Frenette M | title = Evolutionary relationships among salivarius streptococci as inferred from multilocus phylogenies based on 16S rRNA-encoding, recA, secA, and secY gene sequences | journal = BMC Microbiology | volume = 9 | pages = 232 | date = October 2009 | pmid = 19878555 | pmc = 2777182 | doi = 10.1186/1471-2180-9-232 | doi-access = free }}</ref>

===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>

===Medical and diagnostic applications===
Prospective parents can be tested for being [[genetic carrier]]s, or their children might be tested for actually being affected by a [[cystic fibrosis|disease]].<ref name="Saiki1"/> DNA samples for [[Prenatal diagnosis|prenatal testing]] can be obtained by [[amniocentesis]], [[chorionic villus sampling]], or even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis is also essential to [[preimplantation genetic diagnosis]], where individual cells of a developing embryo are tested for mutations.
* PCR can also be used as part of a sensitive test for ''[[tissue typing]]'', vital to [[organ transplantation]]. {{As of|2008|post=,}} there is even a proposal to replace the traditional antibody-based tests for [[blood type]] with PCR-based tests.<ref>{{cite journal | vauthors = Quill E | title = Medicine. Blood-matching goes genetic | journal = Science | volume = 319 | issue = 5869 | pages = 1478–79 | date = March 2008 | pmid = 18339916 | doi = 10.1126/science.319.5869.1478 | s2cid = 36945291 }}</ref>
* Many forms of cancer involve alterations to ''[[oncogene]]s''.  By using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient. PCR permits early diagnosis of [[malignant]] diseases such as [[leukemia]] and [[lymphoma]]s, 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.<ref>{{cite book|last1=Tomar|first1=Rukam|title=Molecular Markers and Plant Biotechnology|date=2010|publisher= New India Publishing Agency|location= Pitman Pura, New Delhi|isbn= 978-93-80235-25-7|page=188}}</ref> PCR is very useful in the medical field since it allows for the isolation and amplification of tumor suppressors. Quantitative PCR for example, can be used to quantify and analyze single cells, as well as recognize DNA, mRNA and protein confirmations and combinations.<ref name="Garibyan, Avashia 1–4"/>

===Infectious disease applications===
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.<ref name=Cai2014>{{cite journal | vauthors = Cai HY, Caswell JL, Prescott JF | title = Nonculture molecular techniques for diagnosis of bacterial disease in animals: a diagnostic laboratory perspective | journal = Veterinary Pathology | volume = 51 | issue = 2 | pages = 341–50 | date = March 2014 | pmid = 24569613 | doi = 10.1177/0300985813511132 | doi-access = free }}</ref> PCR also permits identification of non-cultivatable or slow-growing microorganisms such as [[mycobacterium|mycobacteria]], [[anaerobic organism|anaerobic bacteria]], or [[virus]]es from [[tissue culture]] assays and [[animal model]]s. 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.<ref name= "Cai2014" /><ref>{{cite book |author=Salis AD|year=2009|chapter= Applications in Clinical Microbiology|title=Real-Time PCR: Current Technology and Applications|publisher= Caister Academic Press |isbn= 978-1-904455-39-4}}</ref>

Characterization and detection of infectious disease organisms have been revolutionized by PCR in the following ways:
* The ''human immunodeficiency virus'' (or ''[[HIV]]''), is a difficult target to find and eradicate. The earliest tests for infection relied on the presence of antibodies to the virus circulating in the bloodstream.  However, antibodies don't appear until many weeks after infection, maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the infection don't affect the antibodies.  PCR [[HIV test|tests]] have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells.<ref>{{cite journal | vauthors = Kwok S, Mack DH, Mullis KB, Poiesz B, Ehrlich G, Blair D, Friedman-Kien A, Sninsky JJ | title = Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection | journal = Journal of Virology | volume = 61 | issue = 5 | pages = 1690–94 | date = May 1987 | pmid = 2437321 | pmc = 254157 | doi = 10.1128/jvi.61.5.1690-1694.1987 }}</ref> Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be [[Viral load|quantified]].
* Some disease organisms, such as that for ''[[tuberculosis]]'', are difficult to sample from patients and slow to be [[Tuberculosis diagnosis|grown]] in the laboratory.  PCR-based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient [[Sputum|samples]].  Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy.  The effects of therapy can also be immediately evaluated.
* The spread of a [[pathogen|disease organism]] through populations of [[Domestication#Animals|domestic]] or [[Wildlife|wild]] animals can be monitored by PCR testing.  In many cases, the appearance of new virulent sub-types can be detected and monitored.  The sub-types of an organism that were responsible for [[List of epidemics|earlier epidemics]] can also be determined by PCR analysis.
* Viral DNA can be detected by PCR. The primers used must be specific to the targeted sequences in the DNA of a virus, and 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.<ref name="Cai2014"/> Such early detection may give physicians a significant lead time in treatment. The amount of virus ("[[viral load]]") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below). A variant of PCR ([[Reverse transcription polymerase chain reaction|RT-PCR]]) is used for detecting viral RNA rather than DNA: in this test the enzyme reverse transcriptase is used to generate a DNA sequence which matches the viral RNA; this DNA is then amplified as per the usual PCR method. RT-PCR is widely used to detect the SARS-CoV-2 viral genome.<ref>{{cite web |title=Coronavirus: il viaggio dei test |url= https://www.iss.it/web/guest/primo-piano/-/asset_publisher/o4oGR9qmvUz9/content/id/5269706 |website=Istituto Superiore di Sanità}}</ref>
* Diseases such as pertussis (or [[whooping cough]]) are caused by the bacteria ''[[Bordetella pertussis]]''. This bacteria is marked by a serious acute respiratory infection that affects various animals and humans and has led to the deaths of many young children. The pertussis toxin is a protein exotoxin that binds to cell receptors by two [[Dimer (chemistry)|dimers]] and reacts with different cell types such as T lymphocytes which play a role in cell immunity.<ref>{{Cite book|url= https://www.ncbi.nlm.nih.gov/books/NBK7813/ |title=Medical Microbiology|last1= Finger|first1=Horst|last2=von Koenig|first2= Carl Heinz Wirsing|date=1996|publisher=University of Texas Medical Branch at Galveston |isbn= 978-0-9631172-1-2 |editor-last=Baron|editor-first= Samuel|edition=4th |location= Galveston, TX |pmid= 21413270}}</ref> PCR is an important testing tool that can detect sequences within the gene for the pertussis toxin.  Because PCR has a high sensitivity for the toxin and a rapid turnaround time, it is very efficient for diagnosing pertussis when compared to culture.<ref>{{Cite book |last1= Yeh|first1= Sylvia H.|last2=Mink|first2= ChrisAnna M.|title= Netter's Infectious Diseases|year=2012 |pages= 11–14 |doi= 10.1016/B978-1-4377-0126-5.00003-3 |chapter= Bordetella pertussis and Pertussis (Whooping Cough) |isbn= 978-1-4377-0126-5}}</ref>

===Forensic applications===
The development of PCR-based [[Genetic fingerprinting|genetic]] (or [[DNA fingerprinting|DNA]]) fingerprinting protocols has seen widespread application in [[forensics]]:
* [[File:US Army CID agents at crime scene.jpg|thumb|DNA samples are often taken at crime scenes and analyzed by PCR.]]In its most discriminating form, ''[[genetic fingerprinting]]'' can uniquely discriminate any one person from the entire population of the [[Earth|world]].  Minute samples of DNA can be isolated from a [[O. J. Simpson murder case|crime scene]], and [[Combined DNA Index System|compared]] to that from suspects, or from a [[National DNA database|DNA database]] of earlier evidence or convicts.  Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation.  Evidence from decades-old crimes can be tested, confirming or [[Innocence Project|exonerating]] the people originally convicted.
* Forensic DNA typing has been an effective way of identifying or exonerating criminal suspects due to analysis of evidence discovered at a crime scene. The human genome has many repetitive regions that can be found within gene sequences or in non-coding regions of the genome.  Specifically, up to 40% of human DNA is repetitive.<ref name="Ninfa-2009"/> There are two distinct categories for these repetitive, non-coding regions in the genome. The first category is called variable number tandem repeats (VNTR), which are 10–100 base pairs long and the second category is called short tandem repeats (STR) and these consist of repeated 2–10 base pair sections.  PCR is used to amplify several well-known VNTRs and STRs using primers that flank each of the repetitive regions. The sizes of the fragments obtained from any individual for each of the STRs will indicate which alleles are present. By analyzing several STRs for an individual, a set of alleles for each person will be found that statistically is likely to be unique.<ref name="Ninfa-2009">{{Cite book|title= Fundamental Laboratory Approaches for Biochemistry and Biotechnology|last1= Ninfa |first1=Alexander|last2=Ballou|first2= David|last3=Benore|first3=Marilee |publisher=Wiley|year=2009|isbn= 978-0-470-08766-4|location=United States|pages=408–10}}</ref> Researchers have identified the complete sequence of the human genome. This sequence can be easily accessed through the NCBI website and is used in many real-life applications. For example, the FBI has compiled a set of DNA marker sites used for identification, and these are called the Combined DNA Index System (CODIS) DNA database.<ref name="Ninfa-2009" /> Using this database enables statistical analysis to be used to determine the probability that a DNA sample will match.  PCR is a very powerful and significant analytical tool to use for forensic DNA typing because researchers only need a very small amount of the target DNA to be used for analysis. For example, a single human hair with attached hair follicle has enough DNA to conduct the analysis. Similarly, a few sperm, skin samples from under the fingernails, or a small amount of blood can provide enough DNA for conclusive analysis.<ref name="Ninfa-2009" />
* Less discriminating forms of [[DNA fingerprinting]] can help in ''[[DNA paternity testing]]'', where an individual is matched with their close relatives.  DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children.  Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child.  The actual biological father of a newborn can also be [[DNA paternity testing|confirmed]] (or ruled out).
* The PCR AMGX/AMGY design {{clarify|date=November 2020|reason=Usually, when you say that something not only does one thing, you finish the sentence by describing a second thing that it does.|text=has been shown to not only}} facilitate in amplifying DNA sequences from a very minuscule amount of genome. However it can also be used for real-time sex determination from forensic bone samples. This provides a powerful and effective way to determine gender in forensic cases and ancient specimens.<ref>{{cite journal | vauthors = Alonso A, Martín P, Albarrán C, García P, García O, de Simón LF, García-Hirschfeld J, Sancho M, de La Rúa C, Fernández-Piqueras J | title = Real-Time PCR Designs to Estimate Nuclear and Mitochondrial DNA Copy Number in Forensic and Ancient DNA Studies | journal = Forensic Science International | volume = 139 | issue = 2–3 | pages = 141–49 | date = January 2004 | pmid = 15040907 | doi = 10.1016/j.forsciint.2003.10.008 }}</ref>

===Research applications===
PCR has been applied to many areas of research in molecular genetics:
* PCR allows rapid production of short pieces of DNA, even when not more than the sequence of the two primers is known.  This ability of PCR augments many methods, such as generating ''[[Nucleic acid hybridization|hybridization]] [[Hybridization probe|probes]]'' for [[Southern blot|Southern]] or [[northern blot]] hybridization.  PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, enabling analysis even from very small amounts of starting material.
* The task of ''[[DNA sequencing]]'' can also be assisted by PCR.  Known segments of DNA can easily be produced from a patient with a genetic disease mutation.  Modifications to the amplification technique can extract segments from a completely unknown genome, or can generate just a single strand of an area of interest.
* PCR has numerous applications to the more traditional process of ''[[DNA cloning]]''.  It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities.  Using a single set of 'vector primers', it can also analyze or extract fragments that have already been inserted into vectors.  Some alterations to the PCR protocol can ''generate mutations'' (general or site-directed) of an inserted fragment.
* ''[[Sequence-tagged site]]s'' is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone.  The [[Human Genome Project]] found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories.
* An application of PCR is the [[Phylogeny|phylogenic]] analysis of DNA from ''[[Ancient DNA|ancient sources]]'', such as that found in the recovered bones of [[Neanderthal]]s, from frozen tissues of [[mammoth]]s, or from the brain of Egyptian mummies.<ref name="Schochetman 1988 1154–1157" /> In some cases the highly degraded DNA from these sources might be reassembled during the early stages of amplification.
* A common application of PCR is the study of patterns of ''[[gene expression]]''.  Tissues (or even individual cells) can be analyzed at different stages to see which genes have become active, or which have been switched off.  This application can also use [[quantitative PCR]] to quantitate the actual levels of expression
* The ability of PCR to simultaneously amplify several loci from individual sperm<ref>{{cite journal | vauthors = Boehnke M, Arnheim N, Li H, Collins FS | title = Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm: experimental design considerations | journal = American Journal of Human Genetics | volume = 45 | issue = 1 | pages = 21–32 | date = July 1989 | pmid = 2568090 | pmc = 1683385 }}</ref> has greatly enhanced the more traditional task of ''[[Genetic linkage|genetic mapping]]'' by studying [[chromosomal crossover]]s after [[meiosis]].  Rare crossover events between very close loci have been directly observed by analyzing thousands of individual sperms.  Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all without having to wait (or pay) for the long and laborious processes of fertilization, embryogenesis, etc.
* [[Site-directed mutagenesis]]: PCR can be used to create mutant genes with mutations chosen by scientists at will.  These mutations can be chosen in order to understand how proteins accomplish their functions, and to change or improve protein function.