A strip of eight PCR tubes, each containing a 100 μL reaction mixture
A strip of eight PCR tubes, each containing a 100 μL reaction mixture
Placing a strip of eight PCR tubes into a thermal cycler
Placing a strip of eight PCR tubes into a thermal cycler

The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA,[1] were jointly awarded the Nobel Prize in Chemistry in 1993.

PCR is fundamental to many of the procedures used in genetic testing and research, including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and criminal forensics.[2][3]

The majority of PCR methods rely on thermal cycling. Thermal cycling exposes reactants to repeated cycles of heating and cooling to permit different temperature-dependent reactions—specifically, DNA melting and enzyme-driven DNA replication. PCR employs two main reagents—primers (which are short single strand DNA fragments known as oligonucleotides that are a complementary sequence to the target DNA region) and a DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called nucleic acid denaturation. In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat-susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.[4]

Applications of the technique include DNA cloning for sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based phylogenies, or functional analysis of genes; diagnosis and monitoring of genetic disorders; amplification of ancient DNA;[5] analysis of genetic fingerprints for DNA profiling (for example, in forensic science and parentage testing); and detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases.

Principles

A thermal cycler for PCR
A thermal cycler for PCR
An older, three-temperature thermal cycler for PCR
An older, three-temperature thermal cycler for PCR

PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp) in length, although some techniques allow for amplification of fragments up to 40 kbp.[6] The amount of amplified product is determined by the available substrates in the reaction, which becomes limiting as the reaction progresses.[7]

A basic PCR set-up requires several components and reagents,[8] including:

The reaction is commonly carried out in a volume of 10–200 μL in small reaction tubes (0.2–0.5 mL volumes) in a thermal cycler. 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 Peltier effect, 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 equilibrium. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermal cyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

Typically, PCR consists of a series of 20–40 repeated temperature changes, called thermal cycles, with each cycle commonly consisting of two or three discrete temperature steps (see figure below). The cycling is often preceded by a single temperature step at a very high temperature (>90 °C (194 °F)), 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, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[12] The individual steps common to most PCR methods are as follows:

It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical annealing temperature is about 3–5 °C below the Tm of the primers used. Stable hydrogen bonds between complementary bases are formed only when the primer sequence very closely matches the template sequence. During this step, the polymerase binds to the primer-template hybrid and begins DNA formation.
The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies. The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n, where n is the number of cycles. Thus, a reaction set for 30 cycles results in 230, or 1,073,741,824, copies of the original double-stranded DNA target region.
Schematic drawing of a complete PCR cycle
Ethidium bromide-stained PCR products after gel electrophoresis. 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.
Ethidium bromide-stained PCR products after gel electrophoresis. 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.

To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis may be employed for size separation of the PCR products. The size of the PCR products is determined by comparison with a DNA ladder, a molecular weight marker which contains DNA fragments of known sizes, which runs on the gel alongside the PCR products.

Tucker PCR

Stages

Exponential amplification
Exponential amplification

As with other chemical reactions, the reaction rate and efficiency of PCR are affected by limiting factors. Thus, the entire PCR process can further be divided into three stages based on reaction progress:

Optimization

Main article: PCR optimization

In practice, PCR can fail for various reasons, such as sensitivity or contamination.[17][18] Contamination with extraneous DNA can lead to spurious products and is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[8] For instance, if DNA from a crime scene is analyzed, a single DNA molecule from lab personnel could be amplified and misguide the investigation. Hence the PCR-setup areas is separated from the analysis or purification of other PCR products, disposable plasticware used, and the work surface between reaction setups needs to be thoroughly cleaned.

Specificity can be adjusted by experimental conditions so that no spurious products are generated. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of unspecific products. The usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. For instance, Q5 polymerase is said to be ~280 times less error-prone than Taq polymerase.[19][20] Both the running parameters (e.g. temperature and duration of cycles), or the addition of reagents, such as formamide, may increase the specificity and yield of PCR.[21] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.[22]

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 probes for Southern or 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.

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 E. coli) can be rapidly screened by PCR for correct DNA vector constructs.[23] 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.

Electrophoresis of PCR-amplified DNA fragments: FatherChildMotherThe child has inherited some, but not all, of the fingerprints of each of its parents, giving it a new, unique fingerprint.
Electrophoresis of PCR-amplified DNA fragments:
  1. Father
  2. Child
  3. Mother

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 clocks are used (i.e. the 16S rRNA and recA genes of microorganisms).[24]

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 mummies to the identification of a Russian tsar and the body of English king Richard III.[25]

Quantitative PCR or Real Time PCR (qPCR,[26] not to be confused with 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 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,[27] increasing chances for detection of genes associated with genetic diseases such as cancer.[5] Laboratories use RT-qPCR for the purpose of sensitively measuring gene regulation. The mathematical foundations for the reliable quantification of the PCR[28] and RT-qPCR[29] facilitate the implementation of accurate fitting procedures of experimental data in research, medical, diagnostic and infectious disease applications.[30][31][32][33]

Medical and diagnostic applications

Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease.[2] DNA samples for 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.

Infectious disease applications

PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.[36] PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. 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.[36][37]

Characterization and detection of infectious disease organisms have been revolutionized by PCR in the following ways:

Forensic applications

The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics:

Research applications

PCR has been applied to many areas of research in molecular genetics:

Advantages

PCR has a number of advantages. It is fairly simple to understand and to use, and produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. qRT-PCR shares the same advantages as the PCR, with an added advantage of quantification of the synthesized product. Therefore, it has its uses to analyze alterations of gene expression levels in tumors, microbes, or other disease states.[27]

PCR is a very powerful and practical research tool. The sequencing of unknown etiologies of many diseases are being figured out by the PCR. The technique can help identify the sequence of previously unknown viruses related to those already known and thus give us a better understanding of the disease itself. If the procedure can be further simplified and sensitive non-radiometric detection systems can be developed, the PCR will assume a prominent place in the clinical laboratory for years to come.[16]

Limitations

One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification.[27] This means that, typically, PCR users must know the precise sequence(s) upstream of the target region on each of the two single-stranded templates in order to ensure that the DNA polymerase properly binds to the primer-template hybrids and subsequently generates the entire target region during DNA synthesis.

Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR fragments that are generated.[44]

Another limitation of PCR is that even the smallest amount of contaminating DNA can be amplified, resulting in misleading or ambiguous results. To minimize the chance of contamination, investigators should reserve separate rooms for reagent preparation, the PCR, and analysis of product. Reagents should be dispensed into single-use aliquots. Pipettors with disposable plungers and extra-long pipette tips should be routinely used.[16] It is moreover recommended to ensure that the lab set-up follows a unidirectional workflow. No materials or reagents used in the PCR and analysis rooms should ever be taken into the PCR preparation room without thorough decontamination.[45]

Environmental samples that contain humic acids may inhibit PCR amplification and lead to inaccurate results.

Variations

Main article: Variants of PCR

History

Diagrammatic representation of an example primer pair. The use of primers in an in vitro assay to allow DNA synthesis was a major innovation that allowed the development of PCR.
Diagrammatic representation of an example primer pair. The use of primers in an in vitro assay to allow DNA synthesis was a major innovation that allowed the development of PCR.

Main article: History of polymerase chain reaction

The heat-resistant enzymes that are a key component in polymerase chain reaction were discovered in the 1960s as a product of a microbial life form that lived in the superheated waters of Yellowstone's Mushroom Spring.[79]

A 1971 paper in the Journal of Molecular Biology by Kjell Kleppe and co-workers in the laboratory of H. Gobind Khorana first described a method of using an enzymatic assay to replicate a short DNA template with primers in vitro.[80] However, this early manifestation of the basic PCR principle did not receive much attention at the time and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[81][page needed]

"Baby Blue", a 1986 prototype machine for doing PCR
"Baby Blue", a 1986 prototype machine for doing PCR

When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies, where he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived the idea for PCR while cruising along the Pacific Coast Highway one night in his car.[82] 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 Scientific American, 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."[83] DNA fingerprinting was first used for paternity testing in 1988.[84]

Mullis has credited his use of LSD as integral to his development of PCR: "Would I have invented PCR if I hadn't taken LSD? I seriously doubt it. I could sit on a DNA molecule and watch the polymers go by. I learnt that partly on psychedelic drugs."[85]

Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA,[1] were jointly awarded the Nobel Prize in Chemistry in 1993, seven years after Mullis and his colleagues at Cetus first put his proposal to practice.[86] Mullis's 1985 paper with R. K. Saiki and H. A. Erlich, "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia"—the polymerase chain reaction invention (PCR)—was honored by a Citation for Chemical Breakthrough Award from the Division of History of Chemistry of the American Chemical Society in 2017.[87][2]

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.[2] 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.

The discovery in 1976 of Taq polymerase—a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments[14] such as hot springs—paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[15] thus obviating the need to add new DNA polymerase after each cycle.[3] This allowed an automated thermocycler-based process for DNA amplification.

Patent disputes

The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The Swiss pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992. The last of the commercial PCR patents expired in 2017.[88]

A related patent battle over the Taq polymerase enzyme is still ongoing[as of?] in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on 28 March 2005.[89]

See also

References

  1. ^ a b "The Nobel Prize in Chemistry 1993". NobelPrize.org.
  2. ^ a b c d Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (December 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science. 230 (4732): 1350–54. Bibcode:1985Sci...230.1350S. doi:10.1126/science.2999980. PMID 2999980.
  3. ^ a b Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. (January 1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–91. Bibcode:1988Sci...239..487S. doi:10.1126/science.239.4839.487. PMID 2448875.
  4. ^ Enners, Edward; Porta, Angela R. (2012). "Determining Annealing Temperatures for Polymerase Chain Reaction". The American Biology Teacher. 74 (4): 256–60. doi:10.1525/abt.2012.74.4.9. S2CID 86708426.
  5. ^ a b c d e f Ninfa, Alexander; Ballou, David; Benore, Marilee (2009). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. United States: Wiley. pp. 408–10. ISBN 978-0-470-08766-4.
  6. ^ Cheng S, Fockler C, Barnes WM, Higuchi R (June 1994). "Effective amplification of long targets from cloned inserts and human genomic DNA". Proceedings of the National Academy of Sciences of the United States of America. 91 (12): 5695–99. Bibcode:1994PNAS...91.5695C. doi:10.1073/pnas.91.12.5695. PMC 44063. PMID 8202550.
  7. ^ Carr AC, Moore SD (2012). Lucia A (ed.). "Robust quantification of polymerase chain reactions using global fitting". PLOS ONE. 7 (5): e37640. Bibcode:2012PLoSO...737640C. doi:10.1371/journal.pone.0037640. PMC 3365123. PMID 22701526.
  8. ^ a b Joseph Sambrook & David W. Russel (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 978-0-879-69576-7. Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction
  9. ^ "Polymerase Chain Reaction (PCR)". National Center for Biotechnology Information, U.S. National Library of Medicine.
  10. ^ "PCR". Genetic Science Learning Center, University of Utah.
  11. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (May 2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends in Biotechnology. 22 (5): 253–60. doi:10.1016/j.tibtech.2004.02.011. PMID 15109812.
  12. ^ Rychlik W, Spencer WJ, Rhoads RE (November 1990). "Optimization of the annealing temperature for DNA amplification in vitro". Nucleic Acids Research. 18 (21): 6409–12. doi:10.1093/nar/18.21.6409. PMC 332522. PMID 2243783.
  13. ^ a b Sharkey DJ, Scalice ER, Christy KG, Atwood SM, Daiss JL (May 1994). "Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction". Bio/Technology. 12 (5): 506–09. doi:10.1038/nbt0594-506. PMID 7764710. S2CID 2885453.
  14. ^ a b Chien A, Edgar DB, Trela JM (September 1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". Journal of Bacteriology. 127 (3): 1550–57. doi:10.1128/jb.127.3.1550-1557.1976. PMC 232952. PMID 8432.
  15. ^ a b Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH (May 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". PCR Methods and Applications. 2 (4): 275–87. doi:10.1101/gr.2.4.275. PMID 8324500.
  16. ^ a b c d Schochetman G, Ou CY, Jones WK (December 1988). "Polymerase chain reaction". The Journal of Infectious Diseases. 158 (6): 1154–57. doi:10.1093/infdis/158.6.1154. JSTOR 30137034. PMID 2461996.
  17. ^ Borman, Jon; Schuster, David; Li, Wu-bo; Jessee, Joel; Rashtchian, Ayoub (2000). "PCR from problematic templates" (PDF). Focus. 22 (1): 10. Archived from the original (PDF) on 7 March 2017.
  18. ^ Bogetto, Prachi; Waidne, Lisa; Anderson, Holly (2000). "Helpful tips for PCR" (PDF). Focus. 22 (1): 12. Archived from the original (PDF) on 7 March 2017.
  19. ^ Biolabs, New England. "Q5® High-Fidelity DNA Polymerase | NEB". www.neb.com. Retrieved 4 December 2021.
  20. ^ Sze, Marc A.; Schloss, Patrick D. (2019). "The Impact of DNA Polymerase and Number of Rounds of Amplification in PCR on 16S rRNA Gene Sequence Data". mSphere. 4 (3): e00163–19. doi:10.1128/mSphere.00163-19. PMC 6531881. PMID 31118299.
  21. ^ Sarkar G, Kapelner S, Sommer SS (December 1990). "Formamide can dramatically improve the specificity of PCR". Nucleic Acids Research. 18 (24): 7465. doi:10.1093/nar/18.24.7465. PMC 332902. PMID 2259646.
  22. ^ "Electronic PCR". NCBI – National Center for Biotechnology Information. Retrieved 13 March 2012.
  23. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes". In Kieleczawa J (ed.). DNA Sequencing II: Optimizing Preparation and Cleanup. Jones & Bartlett. pp. 241–57. ISBN 978-0-7637-3383-4.
  24. ^ Pombert JF, Sistek V, Boissinot M, Frenette M (October 2009). "Evolutionary relationships among salivarius streptococci as inferred from multilocus phylogenies based on 16S rRNA-encoding, recA, secA, and secY gene sequences". BMC Microbiology. 9: 232. doi:10.1186/1471-2180-9-232. PMC 2777182. PMID 19878555.
  25. ^ "Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343)". Arizona State University. Archived from the original on 9 October 1997. Retrieved 29 October 2007.
  26. ^ Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. (April 2009). "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments" (PDF). Clinical Chemistry. 55 (4): 611–22. doi:10.1373/clinchem.2008.112797. PMID 19246619.
  27. ^ a b c d Garibyan L, Avashia N (March 2013). "Polymerase chain reaction". The Journal of Investigative Dermatology. 133 (3): 1–4. doi:10.1038/jid.2013.1. PMC 4102308. PMID 23399825.
  28. ^ Schnell, S.; Mendoza, C. (October 1997). "Theoretical Description of the Polymerase Chain Reaction". Journal of Theoretical Biology. 188 (3): 313–18. Bibcode:1997JThBi.188..313S. doi:10.1006/jtbi.1997.0473. PMID 9344735.
  29. ^ Schnell, S.; Mendoza, C. (21 February 1997). "Enzymological Considerations for the Theoretical Description of the Quantitative Competitive Polymerase Chain Reaction (QC-PCR)". Journal of Theoretical Biology. 184 (4): 433–40. Bibcode:1997JThBi.184..433S. doi:10.1006/jtbi.1996.0283. ISSN 0022-5193. PMID 9082073.
  30. ^ Becker, Sven; Böger, Peter; Oehlmann, Ralfh; Ernst, Anneliese (1 November 2000). "PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities". Applied and Environmental Microbiology. 66 (11): 4945–53. Bibcode:2000ApEnM..66.4945B. doi:10.1128/AEM.66.11.4945-4953.2000. ISSN 1098-5336. PMC 92404. PMID 11055948.
  31. ^ Solomon, Anthony W.; Peeling, Rosanna W.; Foster, Allen; Mabey, David C. W. (1 October 2004). "Diagnosis and Assessment of Trachoma". Clinical Microbiology Reviews. 17 (4): 982–1011. doi:10.1128/CMR.17.4.982-1011.2004. ISSN 0893-8512. PMC 523557. PMID 15489358.
  32. ^ Ramzy, Reda M.R. (April 2002). "Recent advances in molecular diagnostic techniques for human lymphatic filariasis and their use in epidemiological research". Transactions of the Royal Society of Tropical Medicine and Hygiene. 96: S225–29. doi:10.1016/S0035-9203(02)90080-5. PMID 12055843.
  33. ^ Sachse, Konrad (2003). "Specificity and performance of diagnostic PCR assays". In Sachse, Konrad; Frey, Joachim (eds.). PCR Detection of Microbial Pathogens. Methods in Molecular Biology. Vol. 216. Totowa, New Jersey: Humana Press. pp. 3–29. doi:10.1385/1-59259-344-5:03. ISBN 978-1-59259-344-6. PMID 12512353. ((cite book)): Missing or empty |title= (help)
  34. ^ Quill E (March 2008). "Medicine. Blood-matching goes genetic". Science. 319 (5869): 1478–79. doi:10.1126/science.319.5869.1478. PMID 18339916. S2CID 36945291.
  35. ^ Tomar, Rukam (2010). Molecular Markers and Plant Biotechnology. Pitman Pura, New Delhi: New India Publishing Agency. p. 188. ISBN 978-93-80235-25-7.
  36. ^ a b c Cai HY, Caswell JL, Prescott JF (March 2014). "Nonculture molecular techniques for diagnosis of bacterial disease in animals: a diagnostic laboratory perspective". Veterinary Pathology. 51 (2): 341–50. doi:10.1177/0300985813511132. PMID 24569613.
  37. ^ Salis AD (2009). "Applications in Clinical Microbiology". Real-Time PCR: Current Technology and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
  38. ^ Kwok S, Mack DH, Mullis KB, Poiesz B, Ehrlich G, Blair D, et al. (May 1987). "Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection". Journal of Virology. 61 (5): 1690–94. doi:10.1128/jvi.61.5.1690-1694.1987. PMC 254157. PMID 2437321.
  39. ^ "Coronavirus: il viaggio dei test". Istituto Superiore di Sanità.
  40. ^ Finger, Horst; von Koenig, Carl Heinz Wirsing (1996). Baron, Samuel (ed.). Medical Microbiology (4th ed.). Galveston, TX: University of Texas Medical Branch at Galveston. ISBN 978-0-9631172-1-2. PMID 21413270.
  41. ^ Yeh, Sylvia H.; Mink, ChrisAnna M. (2012). "Bordetella pertussis and Pertussis (Whooping Cough)". Netter's Infectious Diseases. Netter's Infectious Diseases. pp. 11–14. doi:10.1016/B978-1-4377-0126-5.00003-3. ISBN 978-1-4377-0126-5.
  42. ^ Alonso A, Martín P, Albarrán C, García P, García O, de Simón LF, et al. (January 2004). "Real-Time PCR Designs to Estimate Nuclear and Mitochondrial DNA Copy Number in Forensic and Ancient DNA Studies". Forensic Science International. 139 (2–3): 141–49. doi:10.1016/j.forsciint.2003.10.008. PMID 15040907.
  43. ^ Boehnke M, Arnheim N, Li H, Collins FS (July 1989). "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm: experimental design considerations". American Journal of Human Genetics. 45 (1): 21–32. PMC 1683385. PMID 2568090.
  44. ^ Zhou YH, Zhang XP, Ebright RH (November 1991). "Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase". Nucleic Acids Research. 19 (21): 6052. doi:10.1093/nar/19.21.6052. PMC 329070. PMID 1658751.
  45. ^ Stursberg, Stephanie (2021). "Setting up a PCR lab from scratch". INTEGRA Biosciences.((cite web)): CS1 maint: url-status (link)
  46. ^ Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, et al. (April 1989). "Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)". Nucleic Acids Research. 17 (7): 2503–16. doi:10.1093/nar/17.7.2503. PMC 317639. PMID 2785681.
  47. ^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (October 1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene. 164 (1): 49–53. doi:10.1016/0378-1119(95)00511-4. PMID 7590320.
  48. ^ Innis MA, Myambo KB, Gelfand DH, Brow MA (December 1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proceedings of the National Academy of Sciences of the United States of America. 85 (24): 9436–40. Bibcode:1988PNAS...85.9436I. doi:10.1073/pnas.85.24.9436. PMC 282767. PMID 3200828.
  49. ^ Pierce KE, Wangh LJ (2007). Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells. Methods in Molecular Medicine. Vol. 132. pp. 65–85. doi:10.1007/978-1-59745-298-4_7. ISBN 978-1-58829-578-1. PMID 17876077.
  50. ^ Krishnan M, Ugaz VM, Burns MA (October 2002). "PCR in a Rayleigh-Bénard convection cell". Science. 298 (5594): 793. doi:10.1126/science.298.5594.793. PMID 12399582.
  51. ^ Priye A, Hassan YA, Ugaz VM (November 2013). "Microscale chaotic advection enables robust convective DNA replication". Analytical Chemistry. 85 (21): 10536–41. doi:10.1021/ac402611s. PMID 24083802.
  52. ^ Schwartz JJ, Lee C, Shendure J (September 2012). "Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules". Nature Methods. 9 (9): 913–15. doi:10.1038/nmeth.2137. PMC 3433648. PMID 22886093.
  53. ^ Vincent M, Xu Y, Kong H (August 2004). "Helicase-dependent isothermal DNA amplification". EMBO Reports. 5 (8): 795–800. doi:10.1038/sj.embor.7400200. PMC 1249482. PMID 15247927.
  54. ^ Chou Q, Russell M, Birch DE, Raymond J, Bloch W (April 1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research. 20 (7): 1717–23. doi:10.1093/nar/20.7.1717. PMC 312262. PMID 1579465.
  55. ^ Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert PD, Chenchik A (June 1994). "TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase". BioTechniques. 16 (6): 1134–37. PMID 8074881.
  56. ^ San Millán RM, Martínez-Ballesteros I, Rementeria A, Garaizar J, Bikandi J (December 2013). "Online exercise for the design and simulation of PCR and PCR-RFLP experiments". BMC Research Notes. 6: 513. doi:10.1186/1756-0500-6-513. PMC 4029544. PMID 24314313.
  57. ^ Zietkiewicz E, Rafalski A, Labuda D (March 1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics. 20 (2): 176–83. doi:10.1006/geno.1994.1151. PMID 8020964.
  58. ^ Ochman H, Gerber AS, Hartl DL (November 1988). "Genetic applications of an inverse polymerase chain reaction". Genetics. 120 (3): 621–23. doi:10.1093/genetics/120.3.621. PMC 1203539. PMID 2852134.
  59. ^ Mueller PR, Wold B (November 1989). "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR". Science. 246 (4931): 780–86. Bibcode:1989Sci...246..780M. doi:10.1126/science.2814500. PMID 2814500.
  60. ^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (September 1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proceedings of the National Academy of Sciences of the United States of America. 93 (18): 9821–26. Bibcode:1996PNAS...93.9821H. doi:10.1073/pnas.93.18.9821. PMC 38513. PMID 8790415.
  61. ^ Isenbarger TA, Finney M, Ríos-Velázquez C, Handelsman J, Ruvkun G (February 2008). "Miniprimer PCR, a new lens for viewing the microbial world". Applied and Environmental Microbiology. 74 (3): 840–49. Bibcode:2008ApEnM..74..840I. doi:10.1128/AEM.01933-07. PMC 2227730. PMID 18083877.
  62. ^ Shen C, Yang W, Ji Q, Maki H, Dong A, Zhang Z (November 2009). "NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions". Nanotechnology. 20 (45): 455103. Bibcode:2009Nanot..20S5103S. doi:10.1088/0957-4484/20/45/455103. PMID 19822925. S2CID 3393115.
  63. ^ Shen, Cenchao (2013). "An Overview of Nanoparticle-Assisted Polymerase Chain Reaction Technology". An Overview of Nanoparticle‐Assisted Polymerase Chain Reaction Technology. US: Wiley-Blackwell Publishing Ltd. pp. 97–106. doi:10.1002/9781118451915.ch5. ISBN 978-1-118-45191-5.
  64. ^ Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (April 1989). "Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension". Gene. 77 (1): 61–68. doi:10.1016/0378-1119(89)90359-4. PMID 2744488.
  65. ^ Moller, Simon (2006). PCR: The Basics. US: Taylor & Francis Group. p. 144. ISBN 978-0-415-35547-6.
  66. ^ David F, Turlotte E (November 1998). "[A method of isothermal gene amplification]" [An Isothermal Amplification Method]. Comptes Rendus de l'Académie des Sciences, Série III. 321 (11): 909–14. Bibcode:1998CRASG.321..909D. doi:10.1016/S0764-4469(99)80005-5. PMID 9879470.
  67. ^ Fabrice David (September–October 2002). "Utiliser les propriétés topologiques de l'ADN: une nouvelle arme contre les agents pathogènes" (PDF). Fusion. Archived from the original (PDF) on 28 November 2007.(in French)
  68. ^ Dobosy JR, Rose SD, Beltz KR, Rupp SM, Powers KM, Behlke MA, Walder JA (August 2011). "RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers". BMC Biotechnology. 11: 80. doi:10.1186/1472-6750-11-80. PMC 3224242. PMID 21831278.
  69. ^ Shyamala, V.; Ferro-Luzzi, Ames G. (1993). Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Genome Walking. Methods in Molecular Biology. Vol. 15. pp. 339–48. doi:10.1385/0-89603-244-2:339. ISBN 978-0-89603-244-6. PMID 21400290.
  70. ^ Bing DH, Boles C, Rehman FN, Audeh M, Belmarsh M, Kelley B, Adams CP (1996). "Bridge amplification: a solid phase PCR system for the amplification and detection of allelic differences in single copy genes". Genetic Identity Conference Proceedings, Seventh International Symposium on Human Identification. Archived from the original on 7 May 2001.
  71. ^ Khan Z, Poetter K, Park DJ (April 2008). "Enhanced solid phase PCR: mechanisms to increase priming by solid support primers". Analytical Biochemistry. 375 (2): 391–93. doi:10.1016/j.ab.2008.01.021. PMID 18267099.
  72. ^ Raoult D, Aboudharam G, Crubézy E, Larrouy G, Ludes B, Drancourt M (November 2000). "Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death". Proceedings of the National Academy of Sciences of the United States of America. 97 (23): 12800–03. Bibcode:2000PNAS...9712800R. doi:10.1073/pnas.220225197. PMC 18844. PMID 11058154.
  73. ^ Liu YG, Whittier RF (February 1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics. 25 (3): 674–81. doi:10.1016/0888-7543(95)80010-J. PMID 7759102.
  74. ^ Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (July 1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification". Nucleic Acids Research. 19 (14): 4008. doi:10.1093/nar/19.14.4008. PMC 328507. PMID 1861999.
  75. ^ Myrick KV, Gelbart WM (February 2002). "Universal Fast Walking for direct and versatile determination of flanking sequence". Gene. 284 (1–2): 125–31. doi:10.1016/S0378-1119(02)00384-0. PMID 11891053.
  76. ^ "Full Text – LaNe RAGE: a new tool for genomic DNA flanking sequence determination". www.ejbiotechnology.info.
  77. ^ Park DJ (January 2005). "A new 5' terminal murine GAPDH exon identified using 5'RACE LaNe". Molecular Biotechnology. 29 (1): 39–46. doi:10.1385/MB:29:1:39. PMID 15668518. S2CID 45702164.
  78. ^ Park DJ (April 2004). "3' RACE LaNe: a simple and rapid fully nested PCR method to determine 3'-terminal cDNA sequence". BioTechniques. 36 (4): 586–88, 590. doi:10.2144/04364BM04. PMID 15088375.
  79. ^ "Key ingredient in coronavirus tests comes from Yellowstone's lakes". Science. 31 March 2020. Retrieved 13 May 2020.
  80. ^ Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG (March 1971). "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases". Journal of Molecular Biology. 56 (2): 341–61. doi:10.1016/0022-2836(71)90469-4. PMID 4927950.
  81. ^ Rabinow, Paul (1996). Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press. ISBN 978-0-226-70146-2.
  82. ^ Mullis, Kary (1998). Dancing Naked in the Mind Field. New York: Pantheon Books. ISBN 978-0-679-44255-4.
  83. ^ Mullis KB (April 1990). "The unusual origin of the polymerase chain reaction". Scientific American. 262 (4): 56–61, 64–65. Bibcode:1990SciAm.262d..56M. doi:10.1038/scientificamerican0490-56. PMID 2315679.
  84. ^ Patidar M, Agrawal S, Parveen F, Khare P (2015). "Molecular insights of saliva in solving paternity dispute". Journal of Forensic Dental Sciences. 7 (1): 76–79. doi:10.4103/0975-1475.150325. PMC 4330625. PMID 25709326.
  85. ^ Nichols D, Barker E (2016). "Psychedelics". Pharmacological Reviews. 68 (2): 264–355. doi:10.1124/pr.115.011478. PMC 4813425. PMID 26841800.
  86. ^ "The Nobel Prize in Chemistry 1993". NobelPrize.org.
  87. ^ "Citations for Chemical Breakthrough Awards 2017 Awardees". Division of the History of Chemistry. Retrieved 12 March 2018.
  88. ^ Fore Jr, J.; Wiechers, I. R.; Cook-Deegan, R. (3 July 2006). "The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study". Journal of Biomedical Discovery and Collaboration. 1: 7. doi:10.1186/1747-5333-1-7. PMC 1523369. PMID 16817955.
  89. ^ "Advice on How to Survive the Taq Wars". GEN Genetic Engineering News – Biobusiness Channel. 26 (9). 1 May 2006.