Editing Polymerase chain reaction (section)

===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]]