This page assumes familiarity with the terms and components used in the Polymerase Chain Reaction

Polymerase chain reaction

The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....

 (PCR) process.

The versatility of PCR has led to a large number of variants:

Basic modifications

Often only a small modification needs to be made to the standard PCR protocol to achieve a desired goal:

• Multiplex-PCR uses several pairs of primers annealing to different target sequences. This permits the simultaneous analysis of multiple targets in a single sample. For example, in testing for genetic mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the targets assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent Probe Amplification (or MLPA
  Multiplex ligation-dependent probe amplification
  Multiplex ligation-dependent probe amplification is a variation of the polymerase chain reaction that permits multiple targets to be amplified with only a single primer pair. Each probe consists of a two oligonucleotides which recognise adjacent target sites on the DNA...
  
  ) permits multiple targets to be amplified using only a single pair of primers, avoiding the resolution limitations of multiplex PCR. Multiplex PCR has also been used for analysis of microsatellites and SNPs
  Single nucleotide polymorphism
  A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...
  
  .

• Variable Number of Tandem Repeats (VNTR) PCR targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeat
  Short tandem repeat
  A short tandem repeat in DNA occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 5 base pairs and is typically in the non-coding intron region...
  
  s (or STRs) is the basis for DNA Fingerprinting databases
  National DNA database
  A national DNA database is a government database of DNA profiles which can be used by law enforcement agencies to identify suspects of crimes....
  
   such as CODIS
  Combined DNA Index System
  The Combined DNA Index System is a DNA database funded by the United States Federal Bureau of Investigation . It is a computer system that stores DNA profiles created by federal, state, and local crime laboratories in the United States, with the ability to search the database to assist in the...
  
  .

• Asymmetric PCR preferentially amplifies one strand of the target DNA. It is used in some sequencing
  Sequencing
  In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...
  
   methods and hybridization probing, to generate one DNA strand as product. Thermocycling is carried out as in PCR, but with a limiting amount or leaving out one of the primers. When the limiting primer becomes depleted, replication increases arithmetically
  Linear equation
  A linear equation is an algebraic equation in which each term is either a constant or the product of a constant and a single variable....
  
   through extension of the excess primer. A modification of this process, named Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher Melting temperature (T_m) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction. (Also see Overlap-extension PCR).

• Some modifications are needed to perform long PCR. The original Klenow-based
  Klenow fragment
  right|thumb|450px|Functional domains in the Klenow Fragment and DNA Polymerase I .The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin...
  
   PCR process did not generate products that were larger than about 400 bp. Taq polymerase
  Taq polymerase
  thumb|228px|right|Structure of Taq DNA polymerase bound to a DNA octamerTaq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965...
  
   can however amplify targets of up to several thousand bp long. Since then, modified protocols with Taq enzyme have allowed targets of over 50 kb to be amplified.

• Nested PCR is used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first PCR, one pair of primers is used to generate DNA products, which may contain products amplified from non-target areas. The products from the first PCR are then used as template in a second PCR, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR, but it requires more detailed knowledge of the sequence of the target.

• Quantitative PCR (or Q-PCR) is used to measure the specific amount of target DNA (or RNA) in a sample. By measuring amplification only within the phase of true exponential increase, the amount of measured product more accurately reflects the initial amount of target. Special thermal cyclers are used that monitor the amount of product during the amplification. Quantitative Real-Time PCR (QRT-PCR) methods use fluorescent dyes, such as Sybr Green, or fluorophore
  Fluorophore
  A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
  
  -containing DNA probes, such as TaqMan
  TaqMan
  TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays. The method was first reported in 1991 by researchers at Cetus Corporation, and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied...
  
  , to measure the amount of amplified product as the amplification progresses.

• Hot-start PCR is a technique performed manually by heating the reaction components to the DNA melting temperature (e.g. 95°C) before adding the polymerase. In this way, non-specific amplification at lower temperatures is prevented. Alternatively, specialized reagents inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody
  Antibody
  An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
  
  , or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. 'Hot-start/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures.

• In Touchdown PCR
  Touchdown polymerase chain reaction
  Touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers will avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The...
  
  , the annealing temperature is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard T_m of the primers used, while in the later cycles it is a similar amount below the T_m. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification at the end of the reaction.

• Assembly PCR
  Polymerase cycling assembly
  Polymerase cycling assembly is a method for the assembly of large DNA oligonucleotides from shorter fragments...
  
   (also known as Polymerase Cycling Assembly or PCA) is the synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments, to assemble two or more pieces of DNA into one piece. It involves an initial PCR with primers that have an overlap and a second PCR using the products as the template that generates the final full-length product. This technique may substitute for ligation
  Ligation
  Ligation may refer to:* In molecular biology, the covalent linking of two ends of DNA molecules using DNA ligase* In medicine, the making of a ligature * Chemical ligation, the production of peptides from amino acids...
  
  -based assembly.

• In Colony PCR, bacterial colonies are screened directly by PCR, for example, the screen for correct DNA vector
  Plasmid
  In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
  
   constructs. Colonies are sampled with a sterile pipette tip and a small quantity of cells transferred into a PCR mix. To release the DNA from the cells, the PCR is either started with an extended time at 95°C (when standard polymerase is used), or with a shortened denaturation step at 100°C and special chimeric DNA polymerase.

• The Digital polymerase chain reaction
  Digital polymerase chain reaction
  Digital Polymerase Chain Reaction is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA...
  
   simultaneously amplifies thousands of samples, each in a separate droplet
  Polony
  Polony is a contraction of "polymerase colony," a small colony of DNA.Polonies are discrete clonal amplifications of a single DNA molecule, grown in a gel matrix. This approach greatly improves the signal-to-noise ratio. Polonies can be generated using several techniques that include solid-phase...
  
   within an emulsion.

Pretreatments and extensions

The basic PCR process can sometimes precede or follow another technique:

• RT-PCR (or Reverse Transcription PCR) is used to reverse-transcribe and amplify RNA
  RNA
  Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
  
   to cDNA. PCR is preceded by a reaction using reverse transcriptase
  Reverse transcriptase
  In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
  
  , an enzyme that converts RNA into cDNA. The two reactions may be combined in a tube, with the initial heating step of PCR being used to inactivate the transcriptase. The Tth polymerase (described below) has RT activity, and can carry out the entire reaction. RT-PCR is widely used in expression profiling
  Expression profiling
  In the field of molecular biology, gene expression profiling is the measurement of the activity of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between cells that are actively dividing, or show how the cells react to a...
  
  , which detects the expression of a gene. It can also be used to obtain sequence of an RNA transcript, which may aid the determination of the transcription start
  Transcription (genetics)
  Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
  
   and termination sites (by RACE-PCR) and facilitate mapping of the location of exons and introns in a gene sequence.

• Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' (or adaptors) that are first ligated to fragments of the target DNA. PCR primers that anneal to the linker sequences are then used to amplify the target fragments. This method is deployed for DNA sequencing, genome walking, and DNA footprinting
  DNA footprinting
  DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells....
  
  . A related technique is Amplified fragment length polymorphism
  Amplified fragment length polymorphism
  Amplified Fragment Length Polymorphism PCR is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990s by Keygene, AFLP uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky...
  
  , which generates diagnostic fragments of a genome.

• Methylation-specific PCR (MSP) is used to identify patterns of DNA methylation
  DNA methylation
  DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
  
   at cytosine-guanine (CpG) island
  CpG island
  In genetics, CpG islands or CG islands are genomic regions that contain a high frequency of CpG sites but to date objective definitions for CpG islands are limited. In mammalian genomes, CpG islands are typically 300-3,000 base pairs in length. They are in and near approximately 40% of promoters of...
  
  s in genomic DNA. Target DNA is first treated with sodium bisulfite
  Sodium bisulfite
  Sodium bisulfite is a chemical compound with the chemical formula NaHSO3. Sodium bisulfite is a food additive with E number E222. This salt of bisulfite can be prepared by bubbling sulfur dioxide in a solution of sodium carbonate in water...
  
  , which converts unmethylated cytosine
  Cytosine
  Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
  
   bases to uracil
  Uracil
  Uracil is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine.Uracil is a common and...
  
  , which is complementary to adenosine in PCR primers. Two amplifications are then carried out on the bisulfite-treated DNA: One primer set anneals to DNA with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with uracil (corresponding to unmethylated cytosine). MSP used in Q-PCR provides quantitative information about the methylation state of a given CpG island.

Other modifications

Adjustments of the components in PCR is commonly used for optimal performance:

• The divalent magnesium
  Magnesium
  Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...
  
   ion (Mg⁺⁺) is required for PCR polymerase activity. Lower concentrations Mg⁺⁺ will increase replication fidelity, while higher concentrations will introduce more mutations.

• Denaturants(such as DMSO) can increase amplification specificity by destabilizing non-specific primer binding. Other chemicals, such as glycerol
  Glycerol
  Glycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. The glycerol backbone is central to all lipids...
  
  , are stabilizers for the activity of the polymerase during amplification. Detergents (such as Triton X-100) can prevent polymerase stick to itself or to the walls of the reaction tube.

• DNA polymerases occasionally incorporate mismatch bases into the extending strand. High-fidelity PCR employs enzymes with 3'-5' exonuclease
  Exonuclease
  Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
  
   activity that decreases this rate of mis-incorporation. Examples of enzymes with proofreading activity include Pfu
  Pfu DNA polymerase
  Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions in vivo to replicate the organism's DNA...
  
  ; adjustments of the Mg⁺⁺ and dNTP concentrations may help maximize the number of products that exactly match the original target DNA.

• COLD-PCR
  COLD-PCR
  COLD-PCR is a modified Polymerase Chain Reaction protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA...
  
   (co-amplification at lower denaturation temperature-PCR) is a modified Polymerase Chain Reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA.

Primer modifications

Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich source of modification:

• Normally PCR primers are chosen from an invariant part of the genome, and might be used to amplify a polymorphic area between them. In Allele-specific PCR the opposite is done. At least one of the primers is chosen from a polymorphic area, with the mutations located at (or near) its 3'-end. Under stringent conditions, a mismatched primer will not initiate replication, whereas a matched primer will. The appearance of an amplification product therefore indicates the genotype. (For more information, see SNP genotyping
  SNP genotyping
  SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation...
  
  .)

• InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths. The use of primers from a commonly repeated segment
  Alu sequence
  An Alu element is a short stretch of DNA originally characterized by the action of the Alu restriction endonuclease. Alu elements of different kinds occur in large numbers in primate genomes. In fact, Alu elements are the most abundant mobile elements in the human genome. They are derived from the...
  
   is called Alu-PCR, and can help amplify sequences adjacent (or between) these repeats.

• Primers can also be designed to be 'degenerate' - able to initiate replication from a large number of target locations. Whole genome amplification (or WGA) is a group of procedures that allow amplification to occur at many locations in an unknown genome, and which may only be available in small quantities. Other techniques use degenerate primers that are synthesized using multiple nucleotides at particular positions (the polymerase 'chooses' the correctly matched primers). Also, the primers can be synthesized with the nucleoside analog
  Nucleic acid analogues
  Nucleic acid analogues are compounds structurally similar to naturally occurring RNA and DNA, used in medicine and in molecular biology research....
  
   inosine
  Inosine
  Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring via a β-N9-glycosidic bond....
  
  , which hybridizes to three of the four normal bases. A similar technique can force PCR to perform Site-directed mutagenesis
  Site-directed mutagenesis
  Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
  
  . (also see Overlap extension polymerase chain reaction
  Overlap extension polymerase chain reaction
  The overlap extension polymerase chain reaction is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension PCR...
  
  )

• Normally the primers used in PCR are designed to be fully complementary to the target. However, the polymerase is tolerant to mis-matches away from the 3' end. Tailed-primers include non-complementary sequences at their 5' ends. A common procedure is the use of linker-primers, which ultimately place restriction sites at the ends of the PCR products, facilitating their later insertion into cloning vectors.

• An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector
  Plasmid
  In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
  
  , and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted.

• PCR can easily be modified to produce a labeled product for subsequent use as a hybridization probe. One or both primers might be used in PCR with a radioactive or fluorescent label already attached, or labels might be added after amplification. These labeling methods can be combined with 'asymmetric-PCR' (above) to produce effective hybridization probes.

DNA Polymerases

There are several DNA polymerases that are used in PCR:

• The Klenow fragment
  Klenow fragment
  right|thumb|450px|Functional domains in the Klenow Fragment and DNA Polymerase I .The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin...
  
  , derived from the original DNA Polymerase I from E. coli, was the first enzyme used in PCR. Because of its lack of stability at high temperature, it needs be replenished during each cycle, and therefore is not commonly used in PCR.

• The bacteriophage T4 DNA polymerase was also initially used in PCR. It has a higher fidelity of replication than the Klenow fragment, but is also destroyed by heat.

• The DNA polymerase from Thermus aquaticus
  Thermus aquaticus
  Thermus aquaticus is a species of bacterium that can tolerate high temperatures, one of several thermophilic bacteria that belong to the Deinococcus-Thermus group...
  
   (or Taq
  Taq polymerase
  thumb|228px|right|Structure of Taq DNA polymerase bound to a DNA octamerTaq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965...
  
  ), was the first thermostable polymerase used in PCR, and is still the one most commonly used. The enzyme can be isolated from its native source, or from its cloned gene expressed in E. coli.

• The Stoffel fragment is made from a truncated gene for Taq polymerase and expressed in E. coli. It is lacking 5'-3' exonuclease activity, and may be able to amplify longer targets than the native enzyme.

• Faststart polymerase is a variant of Taq polymerase that requires strong heat activation, thereby avoiding non-specific amplification due to polymerase activity at low temperature (see hot-start PCR above).

• Pfu DNA polymerase
  Pfu DNA polymerase
  Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions in vivo to replicate the organism's DNA...
  
  , isolated from the archean
  Archaea
  The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
  
   Pyrococcus furiosus
  Pyrococcus furiosus
  Pyrococcus furiosus is an extremophilic species of Archaea. It can be classified as a hyperthermophile because it thrives best under extremely high temperatures—higher than those preferred of a thermophile...
  
  , has proofreading activity, and a 5-fold decrease in the error rate of replication compared to Taq. Since errors increase as PCR progresses, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.

• Vent polymerase is an extremely thermostable
  Thermophile
  A thermophile is an organism — a type of extremophile — that thrives at relatively high temperatures, between 45 and 122  °C . Many thermophiles are archaea...
  
   DNA polymerase isolated from Thermococcus litoralis
  Thermococcus litoralis
  Thermococcus litoralis is a species of archaea.-Location:Thermococcus litoralis grows near and around deep-sea smoker vents.-Reproduction:...
  
  .

• Tth polymerase is a thermostable polymerase from Thermus thermophilus
  Thermus thermophilus
  Thermus thermophilus is a Gram negative eubacterium used in a range of biotechnological applications, including as a model organism for genetic manipulation, structural genomics, and systems biology. The bacterium is extremely thermophilic, with an optimal growth temperature of about...
  
  . It has reverse transcriptase activity in the presence of Mn²⁺
  Manganese
  Manganese is a chemical element, designated by the symbol Mn. It has the atomic number 25. It is found as a free element in nature , and in many minerals...
  
   ions, allowing PCR amplification from RNA targets.

Mechanism modifications

Sometimes even the basic mechanism of PCR can be modified:

• Unlike normal PCR, Inverse PCR
  Inverse polymerase chain reaction
  Inverse polymerase chain reaction is a variant of the polymerase chain reaction that is used to amplify DNA with only one known sequence...
  
   allows amplification and sequencing of DNA that surrounds a known sequence. It involves initially subjecting the target DNA to a series of restriction enzyme
  Restriction enzyme
  A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
  
   digestions
  Restriction digest
  A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation...
  
  , and then circularizing the resulting fragments by self ligation. Primers are designed to be extended outward from the known segment, resulting in amplification of the rest of the circle. This is especially useful in identifying sequences to either side of various genomic inserts.

• Similarly, Thermal Asymmetric InterLaced PCR (or TAIL-PCR) is used to isolate unknown sequences flanking a known area of the genome. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures. A 'degenerate' primer is used to amplify in the other direction from the unknown sequence.

Isothermal amplification methods

Some DNA amplification protocols have been developed that may be used alternatively to PCR:

• Helicase-dependent amplification
  Helicase-dependent amplification
  Helicase-dependent amplification is a method for in vitro DNA amplification like the polymerase chain reaction , but that works at constant temperature.- Introduction :...
  
   is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension steps. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

• PAN-AC also uses isothermal conditions for amplification, and may be used to analyze living cells.

• Nicking Enzyme Amplification Reaction
  Nicking enzyme amplification reaction
  Nicking Enzyme Amplification Reaction is a method for in vitro DNA amplification like the polymerase chain reaction . NEAR is isothermal, replicating DNA at a constant temperature using a polymerase to exponentially amplify the DNA at a temperature range of 55 °C to 59 °C.The disadvantage of PCR...
  
   referred to as NEAR, is isothermal, replicating DNA at a constant temperature using a polymerase and nicking enzyme.

• Recombinase Polymerase Amplification (RPA). The method uses a recombinase to specifically pair primers with double-stranded DNA on the basis of homology, thus directing DNA synthesis from defined DNA sequences present in the sample. Presence of the target sequence initiates DNA amplification, and no thermal or chemical melting of DNA is required. The reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels typically within 5–10 minutes. The entire reaction system is stable as a dried formulation and does not need refrigeration. RPA can be used to replace PCR (Polymerase Chain Reaction) in a variety of laboratory applications and users can design their own assays.

Additional reading

• PCR Applications Manual (from Roche Diagnostics).