Editing Polymerase chain reaction (section)

==Principles==
[[File:Primitive PCR machine for scrap.JPG|thumb|upright|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 pair]]s (kbp) in length, although some techniques allow for amplification of fragments up to 40 kbp.<ref>{{cite journal | vauthors = Cheng S, Fockler C, Barnes WM, Higuchi R | title = Effective amplification of long targets from cloned inserts and human genomic DNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 91 | issue = 12 | pages = 5695–99 | date = June 1994 | pmid = 8202550 | pmc = 44063 | doi = 10.1073/pnas.91.12.5695 | bibcode = 1994PNAS...91.5695C | doi-access = free }}</ref> The amount of amplified product is determined by the available substrates in the reaction, which becomes limiting as the reaction progresses.<ref>{{cite journal | vauthors = Carr AC, Moore SD | title = Robust quantification of polymerase chain reactions using global fitting | journal = PLOS ONE| volume = 7 | issue = 5 | pages = e37640 | year = 2012 | pmid = 22701526 | pmc = 3365123 | doi = 10.1371/journal.pone.0037640 | editor1-last = Lucia | bibcode = 2012PLoSO...737640C | editor1-first = Alejandro | doi-access = free }}</ref>

A basic PCR set-up requires several components and reagents,<ref name=molecular_cloning>{{cite book|author1 = Joseph Sambrook|author2 = David W. Russel|year = 2001|title = Molecular Cloning: A Laboratory Manual|edition = third|publisher = Cold Spring Harbor Laboratory Press|location = Cold Spring Harbor, N.Y.|isbn = 978-0-87969-576-7|url-access = registration|url = https://archive.org/details/molecularcloning0000samb_p7p5}} Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction</ref> including:
* a ''DNA template'' that contains the DNA target region to amplify
* a ''[[DNA polymerase]]''; an enzyme that [[polymerization|polymerizes]] new DNA strands; heat-resistant [[Taq polymerase|''Taq'' polymerase]] is especially common,<ref>{{cite web|url=https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/|title=Polymerase Chain Reaction (PCR)|publisher= National Center for Biotechnology Information, U.S. National Library of Medicine}}</ref> as it is more likely to remain intact during the high-temperature DNA denaturation process
* two DNA ''[[Primer (molecular biology)|primers]]'' that are [[Complementarity (molecular biology)|complementary]] to the [[Directionality (molecular biology)|3' (three prime) ends]] of each of the [[Sense and antisense|sense and anti-sense]] strands of the DNA target (DNA polymerase can only bind to and elongate from a double-stranded region of DNA; without primers, there is no double-stranded initiation site at which the polymerase can bind);<ref name="learn.genetics.utah.edu">{{cite web|url= http://learn.genetics.utah.edu/content/labs/pcr/ |title= PCR |publisher=Genetic Science Learning Center, [[University of Utah]]}}</ref> specific primers that are complementary to the DNA target region are selected beforehand, and are often custom-made in a laboratory or purchased from commercial biochemical suppliers
* ''deoxynucleoside triphosphates'', or dNTPs (sometimes called <!-- see discussion on talk page -->"deoxynucleotide triphosphates"; [[nucleotide]]s containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA strand
* a ''[[buffer solution]]'' providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
* ''[[Bivalent (chemistry)|bivalent]] [[cations]]'', typically [[magnesium]] (Mg) or [[manganese]] (Mn) ions; Mg<sup>2+</sup> is the most common, but Mn<sup>2+</sup> can be used for [[PCR mutagenesis|PCR-mediated DNA mutagenesis]], as a higher Mn<sup>2+</sup> concentration increases the error rate during DNA synthesis;<ref>{{cite journal | vauthors = Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI | title = Recent developments in the optimization of thermostable DNA polymerases for efficient applications | journal = Trends in Biotechnology | volume = 22 | issue = 5 | pages = 253–60 | date = May 2004 | pmid = 15109812 | doi = 10.1016/j.tibtech.2004.02.011 }}</ref> and ''monovalent cations'', typically [[potassium]] (K) ions{{Better source needed|date=November 2020|reason=At least some of these assertions about Mg++ and Mn++ need better sources, see the Talk page.}}

The reaction is commonly carried out in a volume of 10–200&nbsp;[[microliter|μL]] in small reaction tubes (0.2–0.5&nbsp;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 a [[Thermoelectric effect#Peltier effect|Peltier device]], which permits both heating and cooling of the block holding the PCR tubes simply by reversing the device's 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.{{citation needed|date=August 2024}}

===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 (>{{convert|90|°C|°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 [[DNA melting|melting temperature]] (''T<sub>m</sub>'') of the primers.<ref>{{cite journal | vauthors = Rychlik W, Spencer WJ, Rhoads RE | title = Optimization of the annealing temperature for DNA amplification in vitro | journal = Nucleic Acids Research | volume = 18 | issue = 21 | pages = 6409–12 | date = November 1990 | pmid = 2243783 | pmc = 332522 | doi = 10.1093/nar/18.21.6409 }}</ref> The individual steps common to most PCR methods are as follows:
* ''Initialization'': This step is only required for DNA polymerases that require heat activation by [[Hot start PCR|hot-start PCR]].<ref name=antibody_hot_start>{{cite journal | vauthors = Sharkey DJ, Scalice ER, Christy KG, Atwood SM, Daiss JL | title = Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction | journal = Bio/Technology | volume = 12 | issue = 5 | pages = 506–09 | date = May 1994 | pmid = 7764710 | doi = 10.1038/nbt0594-506 | s2cid = 2885453 }}</ref> It consists of heating the reaction chamber to a temperature of {{convert|94|–|96|°C|°F}}, or {{convert|98|°C|°F}} if extremely thermostable polymerases are used, which is then held for 1–10 minutes.{{citation needed|date=August 2024}}
* ''[[Denaturation (biochemistry)#Nucleic acid denaturation|Denaturation]]'': This step is the first regular cycling event and consists of heating the reaction chamber to {{convert|94|–|98|°C|°F}} for 20–30 seconds. This causes [[DNA melting]], or denaturation, of the double-stranded DNA template by breaking the [[hydrogen bond]]s between complementary bases, yielding two single-stranded DNA molecules.
* ''[[Annealing (biology)|Annealing]]'': In the next step, the reaction temperature is lowered to {{convert|50|–|65|°C|°F}} for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand.{{citation needed|date=August 2024}}

: 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 [[DNA–DNA hybridization|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&nbsp;°C below the ''T<sub>m</sub>'' 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.{{citation needed|date=August 2024}}
* ''Extension/Elongation'': The temperature at this step depends on the DNA polymerase used; the optimum [[Enzyme|activity]] temperature for the thermostable DNA polymerase of ''Taq'' polymerase is approximately {{convert|75|–|80|°C|°F}},<ref name="Chien et al.">{{cite journal | vauthors = Chien A, Edgar DB, Trela JM | title = Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus | journal = Journal of Bacteriology | volume = 127 | issue = 3 | pages = 1550–57 | date = September 1976 | pmid = 8432 | pmc = 232952 | doi = 10.1128/jb.127.3.1550-1557.1976 }}</ref><ref name="Lawyer et al.">{{cite journal | vauthors = Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH | title = 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 | journal = PCR Methods and Applications | volume = 2 | issue = 4 | pages = 275–87 | date = May 1993 | pmid = 8324500 | doi = 10.1101/gr.2.4.275 | doi-access = free }}</ref> though a temperature of {{convert|72|°C|°F}} is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that is complementary to the template in the [[Directionality (molecular biology)|5'-to-3' direction]], [[condensation reaction|condensing]] the 5'-[[phosphate group]] of the dNTPs with the 3'-[[hydroxy group]] at the end of the nascent (elongating) DNA strand. The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. As a rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute. Under optimal conditions (i.e., if there are no limitations due to limiting substrates or reagents), at each extension/elongation step, the number of DNA target sequences is doubled. With each successive cycle, the original template strands plus all newly generated strands become template strands for the next round of elongation, leading to exponential (geometric) amplification of the specific DNA target region.{{citation needed|date=August 2024}}

: 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 2<sup>n</sup>, where ''n'' is the number of cycles. Thus, a reaction set for 30 cycles results in 2<sup>30</sup>, or {{formatnum:{{#expr:2^30}}}} copies of the original double-stranded DNA target region.
* ''Final elongation'': This single step is optional, but is performed at a temperature of {{convert|70|–|74|°C|°F}} (the temperature range required for optimal activity of most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.
* ''Final hold'': The final step cools the reaction chamber to {{convert|4|–|15|°C|°F}} for an indefinite time, and may be employed for short-term storage of the PCR products.

[[File:Polymerase chain reaction-en.svg|frameless|center|Schematic drawing of a complete PCR cycle|1024x438px]]
[[Image:Roland Gel.JPG|thumb|[[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.
[[File:Tucker PCR.png|center|Tucker PCR]]

===Stages===
[[File:Exponential Amplification.svg|thumb|upright=1.3|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:
* ''Exponential amplification'': At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). After 30 cycles, a single copy of DNA can be increased up to 1,000,000,000 (one billion) copies. In a sense, then, the replication of a discrete strand of DNA is being manipulated in a tube under controlled conditions.<ref name="Schochetman 1988 1154–1157">{{cite journal | vauthors = Schochetman G, Ou CY, Jones WK | title = Polymerase chain reaction | journal = The Journal of Infectious Diseases | volume = 158 | issue = 6 | pages = 1154–57 | date = December 1988 | pmid = 2461996 | doi = 10.1093/infdis/158.6.1154 | jstor = 30137034 }}</ref> The reaction is very sensitive: only minute quantities of DNA must be present.
* ''Leveling off stage'': The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as dNTPs and primers, causes them to become more limited.
* ''Plateau'': No more product accumulates due to exhaustion of reagents and enzyme.