polymerase chain reaction

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A technique for amplifying DNA sequences in vitro by separating the DNA into two strands and incubating it with oligonucleotide primers and DNA polymerase. It can amplify a specific sequence of DNA by as many as one billion times and is important in biotechnology, forensics, medicine, and genetic research.

A process whereby a strand of deoxyribonucleic acid can be cloned millions of times within a few hours. The process can be used to make prenatal diagnoses of genetic diseases and to identify an individual by analysis of a single tissue cell.

The polymerase chain reaction (PCR) is a laboratory technique for "amplifying" a specific DNA sequence. PCR is extremely efficient and sensitive; it can make millions or billions of copies of any specific sequence of DNA, even when the sequence is in a complex mixture. Because of this power, researchers can use it to amplify sequences even if they only have a minute amount of DNA. A single hair root, or a microscopic blood stain left at a crime scene, for example, contains ample DNA for PCR.

PCR has revolutionized the field of molecular biology. It has enabled researchers to perform experiments easily that previously had been unthinkable. Before the mid-1980s, when PCR was developed, molecular biologists had to use laborious and time-consuming methods to identify, clone, and purify DNA sequences they wanted to study. Kary Mullis was awarded the 1993 Nobel Prize in Chemistry for inventing PCR.

PCR is based on the way cells replicate their DNA. During DNA replication, the two strands of each DNA molecule separate, and DNA polymerase, an enzyme, assembles nucleotides to form two new partner strands for each of the original strands. The original strands serve as templates for the new strands. The new strands are assembled such that each nucleotide in the new strand is determined by the corresponding nucleotide in the template strand. The nucleotides adenine (A) and thymine (T) always lie opposite each other, as do cytosine (C) and guanine (G). Because of this base-pairing specificity, each newly synthesized partner strand has the same sequence as the original partner strand, and replication produces two identical copies of the original double-stranded DNA molecule.

In PCR, a DNA sequence that a researcher wants to amplify, called the "target" sequence, undergoes about thirty rounds of replication in a small reaction tube. During each replication cycle, the number of molecules of the target sequence doubles, because the products and templates of one round of replication all become the templates for the next round. After n rounds of replication, 2ⁿ copies of the target sequence are theoretically produced. After thirty cycles, PCR can produce 2³⁰ or more than ten billion copies of a single target DNA sequence. This is called a polymerase chain reaction because DNA polymerase catalyzes a chain reaction of replication.

Designing Primers

To replicate DNA, DNA polymerases require not only a template, but also a primer. A primer is a sequence of single-stranded DNA that "anneals," or binds, to the template by specific base-pairing. An automated apparatus called an oligonucleotide synthesizer, sometimes nickname a "gene machine," can produce primers of any chosen sequence.

Primers for PCR are typically short sequences, around twenty nucleotides long. It is the primers' sequences that are responsible for PCR's enormous specificity. Researchers design primers so they are likely to bind to sequences on either side of the target DNA. They do so by making the primers complementary to the appropriate sequences and by making them long enough that they are unlikely to bind elsewhere.

The longer the primer, the more likely it is that it will be complementary only to the target sequence. Because any single position in a DNA sequence can be occupied by either an A, T, C, or G, there is a one in four chance that any position will contain an A, for example. (This is an approximation, because the nucleotides are not distributed equally or randomly in DNA.) The odds that any specific DNA sequence that is n nucleotides long would be present at a given spot in a DNA sequence is therefore 1 in 4. The chance that a particular twenty-nucleotide sequence (a typical length for a PCR primer) would occur in a given spot at random is less than one in one trillion (10⁻¹²). The human genome has only about three billion (3 × 10⁹) nucleotide pairs, so any twenty-nucleotide-long sequence is very unlikely to occur more than once by chance in the human genome.

Researchers design two primers that will bind to opposite strands of the DNA on either side of the target sequence. They design them to "point" the right way, so that the section of DNA between, not outside of them, is copied. Designing the primers to "point" in the right direction simply requires building them so that their 3′ ends lie toward the target DNA and their 5′ ends lie away from it. The ends of any segment of DNA, including the complete strand, are chemically different.

One end is called the 5′ (pronounced "5-prime") end. The other is called the 3′ end. In DNA replication, nucleotides are always added to the 3′ end of a growing strand of DNA. DNA synthesis is said to proceed in a 5′ to 3′ direction. The two complementary strands of DNA are anti-parallel, which means that they run in opposite directions. The 5′ end of one strand lies next to the 3′ end of the other, as shown in the diagram.

A Typical Pcr Reaction

A typical PCR reaction consists of the following components, mixed together in a solution with a total volume of between 25 and 100 microliters. The solution must include the template DNA, the primers, nucleotides to serve as building blocks for the newly forming DNA, DNA polymerase to catalyze the synthesis, and buffers and salts, usually including magnesium, that are required for optimal activity of the DNA polymerase. The template can be an unpurified mixture of DNA, such as DNA extracted from a swab of cheek cells from a patient or crime suspect.

To perform the PCR reaction, the tube containing the solution is placed into a machine called a DNA thermal cycler. Thermal cyclers are basically programmable heating blocks. They usually contain a thick aluminum block with holes in which PCR reaction tubes can fit snugly. The block can be rapidly cooled or heated to specific temperatures, for specific lengths of time, under programmable computer control. Each cycle in a PCR reaction is controlled by changing the temperature of the block and, therefore, of the reaction mixture.

The first step in PCR is to heat the mixture to a high temperature, usually 94 to 95 °C, for about five minutes. The hydrogen bonds that hold together the two strands of a double helix are broken at these temperatures, and the DNA separates into single strands. This process is termed denaturation.

In the second step, the PCR mixture is cooled to a lower temperature, typically between about 50 °C and 65 °C. This allows the primers to anneal to their specific complementary sequences in the template DNA. The temperature for this step is chosen carefully to be just low enough to allow the primers to bind, but no cooler. A lower annealing temperature might allow the primers to bind to regions in the template DNA that are not perfect complements, which could lead to the amplification of non-specific sequences.

The optimal annealing temperature for a set of primers can be determined by a formula that is based on the nucleotide composition of the primers, but it is often a matter of trial and error to find the best annealing temperature. The annealing step usually takes about fifteen to thirty seconds, an amazingly short time considering that the primers must "scan" through the template DNA to find their proper binding sites.

In the third step, the reaction is heated again, usually to about 72 °C, the temperature at which the DNA polymerase is most active. Most enzymes are destroyed at 72 °C. In the early days of PCR, scientists used a DNA polymerase that was derived from the bacterium Escherichia coli, which itself is most active at human body temperature, 37 °C. But the E. coli polymerase was destroyed at the high temperatures required for the denaturation and annealing steps, and the polymerase therefore had to be added anew to the reaction, during each PCR cycle.

To solve this problem, scientists purified DNA polymerases from microorganisms that live in hot springs or in deep-sea thermal vents. These organisms' enzymes are most active at high temperatures. The most commonly used enzyme for PCR is called Taq DNA polymerase, which was originally purified from the hot-spring bacterium Thermus aquaticus. (Most commercially available preparations today are recombinant versions, produced in engineered E. coli strains.)

At 72 °C, Taq DNA polymerase adds nucleotides to the 3′ ends of annealed primers at the rate of about two thousand nucleotides per minute. Therefore, to amplify a sequence that is one thousand nucleotides long, the primer extension step must last about thirty seconds at 72 °C. By the end of this step, each template strand has a new complementary strand. This completes the first cycle of the PCR reaction.

The cycle can be repeated, at that point, by restarting the denaturation step. In the next cycle, the original two DNA strands will serve again as templates, as will the two newly synthesized strands. In this way, the number of templates has doubled, and it will double again with each successive cycle.

At the end of the reaction, the tube contains DNA fragments that are almost solely copies of the target DNA. The original template DNA mixture is still present, but for the purpose of most applications (with the exception of subsequent PCR experiments), it is present in negligible amounts compared to the PCR product. The amplified DNA can be analyzed by gel electrophoresis, ligated into a cloning vector, labeled for use as a hybridization probe, or used in numerous other experimental procedures.

Contamination in Pcr Reactions

The extreme sensitivity of PCR for amplifying rare DNA sequences is a mixed blessing. Just as PCR can easily amplify any sequence that a researcher wants to amplify, it can also amplify other sequences.

Amplifying a minute amount of DNA isolated from an ancient mosquito preserved in amber, for instance, could be extremely difficult. DNA from other sources could contaminate the sample during every step, including during the recovery of the amber, while researchers are drilling into it, and while the needle is prepared to remove the mosquito tissue. Contamination of the sample by even a single cell from another source can lead to amplification of that DNA, along with or instead of the mosquito's DNA. Especially if there are segments of contaminating DNA that are similar to the target DNA, primers may bind to the wrong segments.

Or consider a human geneticist who has designed a PCR assay to detect a particular genetic disease. Imagine that a positive result, amplification of the disease allele from a patient's DNA sample, would indicate the patient is a carrier for the disease. If even a trace of DNA from a disease carrier contaminates any of the PCR reagents, then assays performed on samples from non-disease carriers are likely to produce the diagnostic PCR amplification product. It isn't difficult to imagine that a lab that routinely performs this PCR assay might have lots of the tell-tale DNA contaminating benches, pipettes, and even lab coats.

Two approaches address the contamination problem. First, laboratory practices for PCR aim to ensure the utmost cleanliness. Whole new industries have been created to produce contamination-resistant supplies, including micropipette tips. The second solution, as in all carefully planned experiments, is to use controls. Negative controls, including mixtures that have not had any template added or that contain a template known to lack the target sequence, are particularly important for PCR experiments.

Pcr Applications and Variations

PCR is such a powerful, easy, and relatively inexpensive technique that it seems that molecular biologists are always looking for ways to use PCR in their research. Every month, scientific journals describe modifications to tailor the basic PCR approach to new applications.

One variation that has proved very fruitful in gene identification is the use of "degenerate primers." Many genes tend to be highly conserved among different species. Homologues, which are genes from different organisms whose protein products have similar functions, tend to have very similar, but not necessarily identical, sequences. The differences in sequence make it challenging to design standard PCR primers to search for homologues.

However, by comparing the DNA sequences of the gene as it occurs in many different species and finding portions of the sequence that are the same in all the species, a researcher can make an educated guess regarding which nucleotides in an unidentified homologue are likely to be identical to those in a known homologue.

The researcher can design a set of "degenerate" PCR primers, which are primers whose nucleotide sequence is fixed only in those positions where the nucleotides are presumed to be known. In the other positions, nucleotides are allowed to incorporate at random. This makes it likely that at least one of the primers will amplify the unknown target. By conducting PCR with degenerate primer sets, and by using primer annealing temperatures that are lower than normal to allow for less-than-perfect base-pairing, a researcher can often amplify a gene in a single experiment, thus isolating the new homologue and allowing it to be sequenced and studied.

Another important variation on PCR is reverse-transcription PCR. This technique involves first copying RNA into DNA molecules, using the enzyme reverse transcriptase, and subsequently using the standard PCR technique to amplify this complementary DNA (cDNA). Because the messenger RNA content of a cell or tissue represents only the genes that are actively being expressed, this technique provides a powerful method of analyzing gene expression.

Bibliography

Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Publishing, 2002.

Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman, 2000.

Micklos, David A., and Greg A. Freyer. DNA Science: A First Course in Recombinant DNA Technology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1990.

Watson, James D., et al. Recombinant DNA, 2nd ed. New York: Scientific AmericanBooks, 1992.

—Paul J. Muhlrad

For more information on polymerase chain reaction, visit Britannica.com.

A technique used to replicate specific sequences of DNA (the chemical containing hereditary information) in cells. In the 1992 winter Olympic Games at Albertville, PCR was used to verify the gender of 557 women athletes. PCR can replicate the male-determining SRY gene on the Y chromosome, enabling it to be detected. The majority of tests can be completed in 24 h and are highly, but not completely, reliable. See also gender verification.

Polymerase chain reaction, or PCR, is a laboratory technique that amplifies or copies any piece of DNA very quickly without using cells. The DNA is incubated in a test tube with a special kind of DNA polymerase, a supply of nucleotides, and short pieces of syntethic single-stranded DNA that serve a primers for DNA synthesis. With automation, PCR can make billions of copies of a particular segment of DNA in a few hours. Each cycle of the PCR procedure takes only about five minutes. At the end of the cycle, the DNA segment-even one with hundreds of base pairs-has been doubled. A PCR machine repeats the cycle over and over. PCR is much faster than the days it takes to clone a piece of DNA by making a recombinant plasmid and letting it replicate within bacteria.

PCR was developed by the biochemist Kary Mullis (1994-) in 1983 while working for Cetus Corporation, a California biotechnology firm. In 1993, Mullis won the Nobel Prize in Chemistry for developing PCR.

The Polymerase Chain Reaction, or PCR, refers to a widely used technique in molecular biology that has become quintessential in many aspects of DNA analysis with broad-based applications in medicine and forensic investigations. PCR is the amplification of specific sequences of genomic DNA, the genetic material found in virtually all living cells. This technology was conceived by the Californian geneticist Kary B. Mullis (1944), who won a Nobel Prize in chemistry in 1993 for developing PCR. It was first applied to basic science research and later revolutionized modern medicine by improving the diagnosis of human diseases through enhanced genetic testing and medical research. More recently, PCR technology has significantly contributed to both domestic and international forensic sciences as well as applications aimed at improving United States homeland security.

PCR requires specialized equipment that is customized to fluctuate between specifically timed temperature variations. Before PCR is performed, DNA must be isolated from peripheral blood, hair follicles, cheek cells, or tissue samples. Isolated DNA is double stranded, meaning that there are two sequences of letters or nucleotide bases (A or adenine, G or guanine, C or cytosine, and T or thymine). The double stranded DNA is held together by complementary base pairings in that A binds to T, C binds to G and vice versa. Therefore, knowing of the sequence of one strand will reveal the sequence of the complementary strand. Amplification is necessary because there are 3.9 billion bases, and although there is a lot of total DNA, there is not enough to properly analyze specific gene or gene segments. Amplification, therefore, makes it possible to obtain ample quantities of specific sequences of DNA to perform a variety of analyses.

PCR requires "primers," or two sequences about 20–25 bases long with one binding to the beginning sequence of interest and the other binding at the end of the same sequence. In order to get the primers to bind to the targeted sequences in the genome, the PCR machines will undergo several cycles at different temperatures. In the first cycle, the DNA is heated to break apart the two strands. The temperature is then reduced so that the primers can bind or anneal to their complementary base sequence in the DNA. Finally, an enzyme called Taq polymerase adds letters from a pool of bases or letters included in the reaction to the position next to the last base of each the primer. Synthesis of one strand of DNA is in the opposite direction of the other. The result is a double stranded DNA sequence. These cycles are repeated several times and amplification of first the DNA sequence in the genome is copied and this copied DNA is re-copied in the next cycle resulting in exponential growth of the specific sequence. Thirty cycles amplified the target DNA between 100,000- to 10,000,000-fold. However, only DNA sequences of 100 to 2000 bases long are ideally suitable for PCR amplification. In this way, a gene of interest or part of the gene can be amplified to quantities that make genetic studies possible.

PCR, therefore, is rapid, inexpensive, and a relatively easy way of producing a large number of copies of a specific DNA sequence. This is particularly advantageous when there is very little or poor quality DNA. RNA, which is converted from DNA into protein, can also be amplified in the same manner as DNA, however, DNA is much more stable and is easier to isolate. Since each individual inherits sequences of DNA that are different from other individuals, the importance of DNA and PCR technology in identifying an individual is exemplified in the courtroom. DNA analysis can be a powerful tool in criminal investigations, especially those classified as homicides, theft, and sexual assault. Physical evidence left at the scene of any crime can be helpful in reconstructing the sequence of events and potentially reveal the criminal. It can also reveal non-paternity if the pattern of DNA in the offspring does not match the pattern of DNA in the assumed father.

Forensic science relies heavily on PCR technology to amplify specific sequences of DNA that will establish a connection between a specific suspect and a crime scene. Amplification of DNA is critical in cases where the source of DNA is minimal or the integrity is compromised. DNA evidence is also a powerful tool that has been used to ultimately prove the innocence of previously convicted individuals. Additionally, DNA can reveal many characteristics that can help forensic scientists and law enforcement officers identify the perpetrator. This is becoming increasingly applicable to national security as well as international intelligence. PCR has revolutionized law enforcement in this way and will continue to enhance the justice system in the future. For example, using complex algorithms and known sequences of DNA, it is possible to analyze the genetic DNA pattern from an unknown person to predict eye color, gender, and even ethnicity.

In 1985 American geneticist Alec Jeffereys, Ph.D. used PCR technology to amplify regions in the human genome that were highly variable. These DNA fragments were comprised of specific sequences that were repeated. The repeat number was found to be highly variable from individual to individual with the exception of identical twins. These DNA fragments could be amplified using PCR and then studied for variable fragment lengths of repeats. This technology was collectively referred as genetic fingerprinting and became widely used. In a highly publicized case called the Narborough Murder Enquiry, criminal investigators were able to identify the perpetrator using DNA fingerprinting.

One of the more recent applications of PCR technology is for improving national security. After the September 11, 2001 terrorist attacks on the United States, the fear of further attacks involving biological weapons increased. Rapid identification of terrorists and specific biological agents using PCR-based methods represents a plausible approach to gathering critical information about these individuals and weapons.

With the identification, characterization, and genetic engineering of viruses, bacteria, and fungi, the likelihood of strategic, harmful applications involving these organisms is growing. Biological species that represent a serious health risk to humans have been used as weapons for years. These risks can include the U.S. agricultural economy, food supplies, and the environment. To combat bioterrorism, President George W. Bush in February 2002 called for a budget increase to $5.9 billion for Homeland Security directed towards protecting against bioterrorist attacks. The creation of a national database that catalogs pathogens and individuals that are authorized to study these pathogens is also of ongoing concern. A benefit of these databases would be to identify the genetically engineered pathogens used as biological weapons (allowing quick access to specific medical treatment protocols) and to potentially link pathogens to the bioterrorists that developed them.

PCR technology can also be employed to identify the specific disease-causing microorganism. The U.S. Postal Service is working in conjunction with the biotech industry on initiatives to develop intelligent mail. Using PCR to identify anthrax, for example, is one way to quickly ascertain the nature of the contaminated mail or screen high-risk mail. This technology was the government's primary weapon against mail deemed unsuitable for circulation since irradiation provided a limited, unsubstantial solution and often damaged the mail. This high-tech strategy for mail surveillance can be particularly useful by sucking out air samples from the mail and testing for specific molecular signatures using PCR to detect a possible biological contaminant.

Defending against bioterrorism after the September 11 attacks includes developing advances in biological detection instrumentation. In conjunction with the Centers for Disease Control and Prevention (CDC), Lawrence Livermore National Laboratory and its sister laboratory at Los Alamos are currently developing DNA profiles of the most threatening pathogens such as anthrax and the plague using PCR technology. Biodetection instrumentation for genetic profiling has led to the miniaturization and subsequently the portability of DNA analytical devices, particularly for PCR. Forensic scientists and criminologists also benefit from mobile PCR machines by bringing the science to the scene of the crime leading to more rapid crime-solving capabilities.

Security at the 2002 Winter Olympic Games in Salt Lake City was led by The Biological Aerosol Sentry and Information System (BASIS). Miniaturized PCR machines called Smart Cyclers developed by a company called Cepheid were used at the field laboratory operation set up by BASIS. The purpose was to prepare for a bioterrorism threat by having appropriate and rapid biological sample identification to allow for accurate bioterrorist assessment and validation so that the proper responses could be executed.

Recent concerns over genetic engineering of agricultural food products and the potential risks to food safety have prompted studies investigating the molecular signatures of crops using PCR. A study in the scientific journal Nature revealed that genetically manipulated DNA from industrial produced maize had been introduced into corn fields in Oaxaca, Mexico. Although the ramifications to health and food safety are unknown and most likely benign, surveillance of crops using PCR is a formidable approach in the implementation of security measures to help protect against harmful pathogenic contaminations that can threaten food safety. As the cost and use of PCR are eased and as the collection of databases with recognizable DNA profiles of various microorganisms is increased, the utility of this technology in human and food safety will be greatly improved.

Further Reading

Books

Friedman, J., F. Dill, M. Hayden, and B. McGillivray. Genetics. N.P.: Williams & Wilkins, 1996.

Lodish, J., D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell. Molecular Cell Biology. New York: Scientific American Books, 1995.

Periodicals

Jeffereys, A. J. "Hypervariable 'minisatellite' regions in human DNA." Nature no. 314 (1987): 67–73.

Mullis, K. B., and F. A. Faloona. "Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction." Methods in Enzymology no. 155 (1987): 335–350.

Nakamura, Y., M. Leppert, P. O'Connell, et al. "Variable number tandem repeat (VNTR) markers for human gene mapping." Science no. 237 (1987): 1616–1622.

Quist, D., and I. H. Chapela. "Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico." Nature no. 414 (2001): 541–3.

Wong, Z., V. Wilson, A. J. Jeffereys, et al. "Cloning a selected fragment from a human DNA 'fingerprint': isolation of an extremely polymorphic minisatellite." Nucleic Acids no. 14 (1986): 4605–616.

Wyman, A. R. and R. White. "A highly polymorphic locus in human DNA." PNAS no. 77 (1980): 6754–6758.

Electronic

Access Excellence. "Kary B. Mullins." The National Health Museum. March, 2002. <http://www.accessexcellence.org/AB/BC/Kary_B_Mullis.html> (December 13, 2002).

Access Excellence. "PCR Technology." Connie Veilleax. July 8, 2002. <http://www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html> (December 16, 2002).

Center for strategic and international solutions. "New Technology Counters Bioterrorism Threat, Policy Issues." CSIS. Fall, 2002. <http://www.csis.org/pubs/prospectus/02fall_bacastow.htm> (December 11, 2002).

Edvotek. "Biotechnology: DNA Fingerprinting for Forensics and Paternity." 2001. <http://www.edvotek.com/experiments/biotech/04/334.html> (December 12, 2002).

Government Security. "Who are you?" Technology solutions in defense of the homeland. July 22, 2002. <http://govtsecurity.securitysolutions.com> (December 15,2002).

Kari Sable Burns. "Green River Killer." True Crimes. 2002. <http://www.karisable.com/greenriverdnatime.htm> (December 15, 2002).

United States Congress 107th Congress 2nd Session. "Technology assessment in the war on terrorism and homeland security: the role of OTA" Committee Print. April, 2002. <http://www.fas.org/irp/congress/2002_hr/ota.html> (December 15, 2002).

Westburg. "Human Diagnostics: forensics." Cambridge Molecular Diagnostics. 2001. <http://www.westburg.nl/htm/md/hd_forensics.htm> (December 15, 2002).

"PCR" redirects here. For other uses, see PCR (disambiguation).

A strip of eight PCR tubes, each containing a 100 μl reaction

In molecular biology, the polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as the template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Developed in 1984 by Kary Mullis,^[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.^[2][3] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.^[4]

Contents 1 PCR principles and procedure 1.1 Procedure 2 PCR stages 2.1 PCR optimization 3 Application of PCR 3.1 Isolation of genomic DNA 3.2 Amplification and quantitation of DNA 3.3 PCR in diagnosis of diseases 4 Variations on the basic PCR technique 5 History 5.1 Patent wars 6 References 7 External links

PCR principles and procedure

Figure 1a: An old thermal cycler for PCR

Figure 1b: An older model three-temperature thermal cycler for PCR

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.^[5]

A basic PCR set up requires several components and reagents.^[6] These components include:

• DNA template that contains the DNA region (target) to be amplified.
• Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
• Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
• Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
• Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
• Divalent cations, magnesium or manganese ions; generally Mg²⁺ is used, but Mn²⁺ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn²⁺ concentration increases the error rate during DNA synthesis^[7]
• Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction 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 equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.

The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), 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. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.^[8]

• Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.^[9]

• Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes separation of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

• Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

• Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,^[10][11] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

• Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

• Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

Figure 3: 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 generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).

PCR stages

The PCR process can be divided into three stages:

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.^[12]

Levelling 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 limiting.

Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

PCR optimization

Main article: PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.^[13][14] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.^[6] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.

Application of PCR

Isolation of genomic DNA

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, 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 or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs^[15]. 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.

Some PCR 'fingerprints' 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.

Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

Amplification and quantitation of DNA

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.^[16]

Quantitative 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. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

• See also Use of DNA in forensic entomology

PCR in diagnosis of diseases

PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.^{[citation needed]} PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.^{[citation needed]}

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.^{[citation needed]}

Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).

Variations on the basic PCR technique

Main article: Variants of PCR

• Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.^[17] See SNP genotyping for more information.

• Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.^[18]

• Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.^[19] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.^[20]

• Helicase-dependent amplification: This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.^[21]

• Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase.^[22] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody^[9] 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 instantly activated at elongation temperature.

• Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.^[23]

• Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.^[24]

• Ligation-mediated PCR: This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.^[25]

• Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,^[26] and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

• Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that defined two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.^[27] By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.

• Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).

• Multiplex-PCR: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

• Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

• Overlap-extension PCR: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.

• Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.

• RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.

• Single-cell PCR:

• Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR^[28] (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).

• TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. 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.^[29]

• Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5˚C) above the T_m of the primers used, while at the later cycles, it is a few degrees (3-5˚C) below the primer T_m. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.^[30]

• PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.^[31][32]

• Universal Fast Walking: this method allows genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') ^[33] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends) ^[34], 5'RACE LaNe ^[35] and 3'RACE LaNe ^[36].

History

Main article: History of polymerase chain reaction

A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro.^[37] However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.^[38]

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (>195 °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, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F)) environments^[10]) 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,^[11] thus obviating the need to add new DNA polymerase after each cycle^[3]. This allowed an automated thermocycler-based process for DNA amplification.

When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car.^[39] 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."^[40] He was awarded the Nobel Prize in Chemistry in 1993 for his invention,^[4] seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle. (see main article: Kary Mullis)

Patent wars

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 pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.

A related patent battle over the Taq polymerase enzyme is still ongoing 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 March 28, 2005^[41]

References

1. ^ Bartlett & Stirling (2003)—A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6
2. ^ ^{a b} Saiki, RK; Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (December 20 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350–4. doi:10.1126/science.2999980. PMID 2999980. http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/034. 
3. ^ ^{a b} Saiki, RK; Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239: 487–91. doi:10.1126/science.2448875. PMID 2448875. http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009. 
4. ^ ^{a b} Karry Mullis Nobel Lecture, December 8, 1993
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External links

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• PCR Virtual Lab
• US Patent for PCR
• Step-through animation of PCR - Cold Spring Harbor Laboratory Adobe Flash required
• PCR at Home - performing PCRs with low-cost household materials Scientific American

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