polymerase chain reaction
Polymerase Chain Reaction
Polymerase Chain Reaction
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.
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.
Paul J. Muhlrad
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.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
█ BRYAN R. COBB
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:
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.
Jeffereys, A. J. "Hypervariable 'minisatellite' regions in human DNA." Nature no. 314 (1987): 67–73.
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.
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).
Anthrax, Terrorist Use as a Biological Weapon
Biochemical Assassination Weapons
Bio-Engineered Tissue Constructs
Biological and Toxin Weapons Convention
Biological Warfare, Advanced Diagnostics
Biological Weapons, Genetic Identification
CDC (United States Centers for Disease Control and Prevention)
Chemical and Biological Defense Information Analysis Center (CBIAC)
Microbiology: Applications to Espionage, Intelligence and Security
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
PCR (polymerase chain reaction) is a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase, are used to amplify the number of copies of a target DNA sequence by more than 106 times in a few hours. American molecular biologist Kary Mullis developed the idea of PCR in the 1970s. For his ingenious invention, he was awarded the 1993 Nobel Prize in physiology or medicine.
The extraction of DNA polymerase from thermophilic bacteria allowed major advances in PCR technology.
PCR amplification of DNA is like any DNA replication by DNA polymerase in vivo (in living cells). The difference is that PCR produces DNA in a test tube. For a PCR to happen, four components are necessary: template, primer, deoxyribonecleotides (adenine, thymine, cytosine, guanine), and DNA polymerase. In addition, part of the sequence of the targeted DNA has to be known in order to design the according primers. In the first step, the targeted double stranded DNA is heated to over 194°F (90°C) for denaturation. During this process, two strands of the targeted DNA are separated from each other. Each strand is capable of being a template. The second step is carried out around 122°F (50°C). At this lowered temperature, the two primers anneal to their complementary sequence on each template. The DNA polymerase then extends the primer using the provided nucleotides. As a result, at the end of each cycle, the numbers of DNA molecules double.
PCR was carried out manually in incubators of different temperatures for each step until the discovery of DNA polymerase from thermophilic bacteria. The bacterium Thermus aquaticus was found in Yellow Stone National Park. This bacterium lives in the hot springs at 203°F (95°C). The DNA polymerase from T. aquaticus keeps its activity at above 203°F (95°C) for many hours. Several additional heat-resistant DNA polymerases have also now been identified.
Genetic engineered heat resistant DNA polymerases, that have proofreading functions and make fewer mutations in the amplified DNA products, are available commercially. PCR reactions are now carried out in different thermocyclers. Thermocyclers are designed to change temperatures automatically. Researchers set the temperatures and the time, and at the end of the procedure take the test tube out of the machine.
The invention of PCR was revolutionary to molecular biology . PCR is valuable to researchers because it allows them to multiply the quantity of a unique DNA sequence to a large and workable amount in a very short time. Researchers in the Human Genome Project are using PCR to look for markers in cloned DNA segments and to order DNA fragments in libraries. Molecular biologists use PCR to cloning DNA. PCR is also used to produce biotin or other chemical-labeled probes. These probes are used in nucleic acid hybridization, in situ hybridization and other molecular biology procedures.
PCR, coupled with fluorescence techniques and computer technology, allows the real time amplification of DNA. This enables quantitative detection of DNA molecules that exist in minute amounts. PCR is also used widely in clinical tests. Today, it has become routine to use PCR in the diagnosis of infectious diseases such AIDS .
See also Chromosomes, eukaryotic; Chromosomes, prokaryotic; DNA (Deoxyribonucleic acid); DNA chips and micro arrays; DNA hybridization; Immunogenetics; Laboratory techniques in immunology; Laboratory techniques in microbiology; Molecular biology and molecular genetics
Polymerase Chain Reaction
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is used to make millions of copies of a section of deoxyribonucleic acid (DNA ). Until the 1980s obtaining numerous copies of a section of DNA took one to two weeks and required isolation of the DNA, cloning the DNA into a viral or plasmid vector, growing the cloned DNA using living host cells, usually bacteria, and finally isolating the DNA again. With PCR a scientist can produce thirty million copies of a DNA section in a test tube overnight. In a series of early articles, PCR's inventor, Kary Mullis, described just how valuable he believed this tool would be. He could not have been more correct: PCR is now a mainstay of molecular biology. In 1993 Mullis received the Nobel Prize in chemistry in recognition of his discovery.
How PCR Works
PCR repeats the synthesis of a DNA segment through twenty-five to thirty cycles. The power of the technique resides in its ability to copy the products of each previous cycle along with the original sample. This results in exponential growth in the amount of product, as illustrated in Figure 1. The first stage or round of the process only involves copying the original sample. First, the DNA is separated into its two strands, and each strand acts as an instruction directing a new segment. The two products each contain one original strand and one new one. In the second round of the process, both of the DNA molecules manufactured in stage one are separated and each of the four strands is copied, producing four double-stranded products. The number of DNA segments doubles during each round. Twenty-five rounds can create millions of new segments from each original DNA.
To separate the double-stranded DNA products, the reaction is heated to near boiling, 95°C, for a few minutes. Originally, the catalyst, DNA polymerase, was killed by this treatment and manually replaced after each round. In 1988 Randall Saiki and coworkers dramatically improved the original concept of PCR by using a heat-stable catalyst, taq polymerase, isolated from the bacteria, Thermis aquaticus, found in hot springs. This catalyst survives the high temperature step and can start the next round of synthesis when cooled to the start temperature.
The specificity of PCR derives from the primers. Primers are short pieces of single-stranded DNA that are the starting point of the new DNA strands. The primers recognize the target DNA segment in the sample and
bind to the target. Taq polymerase uses the primer as the beginning of the new product strand. Taq polymerase cannot synthesize DNA without a primer at the beginning, so the primers and not the catalyst determine what segment of the sample is copied.
Applications of PCR
Originally, PCR was employed to produce usable amounts of small- and medium-sized DNA segments. Many other important techniques quickly developed. PCR is used to identify bacteria and virus infections. It is not only very sensitive but is also selective enough to distinguish closely related strains. For example, this procedure has been adapted to detect genetically modified crops to help ensure they are utilized only in approved ways.
A widely known application of PCR is DNA fingerprinting. Certain regions of the human chromosome have short sections that are repeated up to several hundred times. Different individuals have different numbers of repeats in each section. If several sections are tested, a unique pattern is observed. Initially, these regions were cut out of sample DNA and tested for size. PCR allows the variable regions to be copied millions of times, greatly increasing the sensitivity and speed of the technique. Because of the technique's sensitivity, extreme care must be taken to avoid any contamination of the samples. Forensic scientists continue to make even greater use of this method.
One of the latest applications of PCR is the DNA microarray or gene chip. It allows medical personnel to test a cancer cell before and after chemotherapy with different drugs. If two patterns look the same, the two drugs administered are likely working on the same pathway and may not be as useful as treatment with two drugs that work by different pathways.
see also Deoxyribonucleic Acid (DNA); Forensic Chemistry.
Erlich, Henry (1995). "PCR Technology." In Molecular Biology and Biotechnology, ed. Robert Meyers. New York: Wiley-VCH.
Guyer, R., and Koshland, Daniel (1989). "The Molecule of the Year." Science 246: 1543.
Joyce, G. (1992). "Directed Molecular Evolution." Scientific American 267: 90.
Mullis, Kary (1990). "The Unusual Origin of the Polymerase Chain Reaction." Scientific American 262: 56.
Polymerase Chain Reaction
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a process that allows one to make in a short amount of time many copies of a particular deoxyribonucleic acid (DNA) sequence. It was developed in 1985 by Kary Mullis and has provided scientists in diverse fields with a powerful tool for DNA amplification, analysis, and manipulation.
The technique involves repeated heating and cooling cycles and requires the DNA polymerase (known as Taq ) from the organism Thermus aquaticus ; these cycles are usually controlled by a machine known as a thermal cycler. Since high temperatures usually inactivate most enzymes , the DNA polymerase used in this procedure is isolated from a heat-tolerant bacterium.
The high temperature (90 to 92 degrees Celsius [194 to 197 degrees Fahrenheit]) disrupts the hydrogen bonds between the bases of DNA and results in the separation of the two DNA strands. Since all enzymes that duplicate DNA require short sequences known as primers , the priming sequences for the desired region are added and the temperature is then lowered. The primers anneal with sequences on each strand of the DNA, and Taq then uses added nucleotides to synthesize a new strand of DNA that is attached to the primers and complementary to each of the original strands.
The sample is then heated again to separate the strands and liberate the primer, and the sample is again cooled to allow another round of DNA duplication. Each round takes only a few minutes, and double the number of DNA strands each time. The process is repeated many more times with the result that thousands, and even millions, of copies of a particular DNA sequence are produced within a few hours. The first two or three rounds of duplication produce DNA molecules of various lengths, but the final rounds produce identically sized molecules.
While early PCR techniques were limited to copying up to 1,000 base pairs , methods developed in the late 1990s have extended the limit up to 10,000 base pairs or more. The procedure will copy any DNA molecule and, consequently, the scientist must make certain that no extraneous DNA is present in the reaction tube.
PCR has allowed people to produce large amounts of DNA from a variety of sources: blood or semen from a crime scene; single embryonic cells for prenatal diagnosis; frozen ancient mammals; cells infected with viruses such as HIV (human immunodeficiency virus).
see also Archaea; Clone; DNA Sequencing; Forensic DNA Analysis; Replication
William R. Wellnitz
Campbell, Neil A., Jane B. Reece, and Lawrence G. Mitchell. Biology, 5th ed. Menlo Park, CA: Benjamin Cummings, 1999.