PCR

PCR (polymerase chain reaction) is a method used by scientists to increase the amount of purified DNA in a sample. It is a highly specific procedure that amplifies one particular gene from within a large sample of undesirable DNA, DNA that the scientist does not wish to replicate. Before PCR, it was very difficult and time consuming to obtain particular fragments of DNA from a sample, and practically impossible to amplify, produce many copies of, that fragment. With PCR, scientists can copy a specific stretch of DNA billions of times in a few hours.

PCR was invented in 1983 by American biochemist Kary B. Mullis, who received the 1993 Nobel Prize for chemistry (with Canadian biochemist Michael Smith) in recognition of this inestimable contribution to science. Mullis invented PCR while working for the Cetus Corporation, a biotechnology firm located in California. His discovery proved so essential to biological research that when Cetus closed down in 1991, the pharmaceutical company Hoffman-La Roche purchased the PCR patent for $300 million.

DNA Replication

Under most natural conditions, DNA exists in the form of two entwined single strands, and each strand is formed of smaller molecules called nucleotides . The word "polymerase" in the name polymerase chain reaction comes from the term "polymer," which refers to any large molecule composed of many smaller molecules. Thus DNA is a polymer of nucleotides. A polymerase is an enzyme that pieces together polymers from the smaller molecules. The strands of DNA must be separated before they can be copied, because the important information is contained along the center of the molecule, where the two strands are attached. Cells naturally synthesize new DNA in their nucleus through a process known as replication. During replication, the two parent strands unwind and separate and a new single daughter strand is built upon each of the existing parent strands. The two identical molecules of DNA that result from replication each contain one parent and one daughter strand. This is called semiconservative replication.

Scientists can find a particular gene (a sequence of nucleotides that encodes a unique protein) within a large sample of DNA by looking for the nucleotide sequences specific to that gene. The four basic nucleotides are adenine (A), guanine (G), thymine (T), and cytosine (C). An A on one strand always binds to a T on the other, and a G on one strand always binds to a C on the other. This makes it easy to determine the sequence of one strand of DNA when the sequence of the other strand is known. The beginnings and ends of all genes are defined by short sequences of three nucleotides called codons . The beginnings are marked by a "start" codon, and the ends by a "stop" codon. Special enzymes in the cell recognize the codons. These enzymes always begin at the start codon and end at the stop codon. Scientists can identify a particular gene by looking at the nucleotide sequence, and locate the place on the DNA where that gene begins by finding the start codon.

PCR Method

Almost any gene encoding almost any protein can be amplified using PCR. To replicate DNA in a laboratory environment, certain natural conditions must be reproduced. The necessary components are simple—the DNA to be replicated, DNA polymerase, primers complementary to both strands of DNA for that gene, and a mixture of the four nucleotides. DNA polymerase "reads" the nucleotide sequence and adds the correct nucleotides to the parent strand, thereby forming a complementary daughter strand. DNA polymerase can only build off a template, and it can add nucleotides only one by one, in one direction. DNA polymerase cannot begin without a primer, a short nucleotide sequence attached at one end of the DNA. For PCR, the primers must be present in very large quantities to increase the likelihood of replication. In a cell, a special enzyme builds primers for DNA, but the process is not specific for any one gene. By using synthetic primers that are complementary to the gene they want to replicate, scientists can replicate only that gene and not the remaining DNA.

PCR can be conducted with as little as one fragment of DNA in solution. By applying a high heat to the DNA solution, the bonds between the parent strands are broken, and the strands float apart. This process is called denaturing the DNA. In the first cycle of PCR amplification, the DNA is denatured, and the primers are added to the solution. The temperature is then lowered so that the primers can bind to the denatured strands. If the temperature is not lowered, the primers will not be able to function. After this, the temperature is raised slightly so that DNA polymerase can bind to the primer. Once it binds, the polymerase pulls nucleotides from the solution and adds them to the template parent strand to form a daughter strand. The polymerase drops away from the newly synthesized DNA at the stop codon. This procedure creates two new molecules of DNA, which contain only the gene the scientist wants to replicate.

The first cycle of PCR generates twice the number of DNA molecules for the gene than there were in the original solution. Additional cycles are needed to greatly increase this number. The second cycle is very similar to the first—the DNA in solution is denatured in the presence of primers, which bind to the parent strands as they cool; polymerase builds the daughter strand with nucleotides from the solution; and the reaction completes itself. The result of this second cycle is that there are now four DNA molecules encoding the gene for every one original DNA molecule used. The cycles are repeated until the desired amount of DNA is attained. Scientists calculate the number of DNA molecules resulting from PCR amplification by employing the formula a ^*2ⁿ, where a is the number of original molecules of DNA, and n is the number of cycles of PCR. When enough DNA is made, the scientist stops the reaction.

The DNA polymerase used for PCR must be able to function at a very high temperature, because a high temperature is needed to keep the parent strands apart so the daughter strands can be built upon them. Most DNA polymerases in nature cannot function at high temperatures, so PCR uses a polymerase found in certain archaebacteria that live in hot springs, where the water temperatures are often well above 90°C (194°F). The most commonly used polymerase is called Taq polymerase, because it was originally isolated from the archaebacterium Thermus aquaticus.

Applications of PCR

PCR is very useful for creating a large quantity of DNA from a very small initial sample. Applications of PCR can be used to identify a particular individual or even to map out the evolutionary history of a species. These applications are based on the concept that some DNA is unique to a particular individual, some genes are unique to a particular species, and certain genes are shared by all organisms. The unique DNA makes it possible to determine the exact individual from which a strand of DNA came, which is why PCR is used by forensic scientists to test skin cells and hair follicles found at crime scenes. Assuming that the DNA belongs to the person who carried out the crime, that person can be identified from among a group of suspects. PCR is used by archaeologists to determine the identity of ancient human remains and unidentifiable mummies, and by paleontologists to examine how the genome of an organism has changed over the course of evolution. PCR can also used to test the relatedness of different species when body characteristics alone do not provide enough evidence. For example, PCR analysis revealed that red panda bears are more closely related to raccoons than to greater panda bears, a distinction that had previously been impossible to determine.

Other applications of PCR take advantage of its ability to accumulate large amounts of DNA to conduct statistically significant research experiments. The technique is often used in medical research, for example to amplify the DNA of a virus, such as HIV, to understand how it infects humans, or to replicate the DNA of a hormone , such as insulin, to understand how it functions. The biomedical industry relies on PCR for identifying viral and bacterial infections, especially for detecting infections like AIDS and leprosy in their early stages. PCR can also be used to detect hereditary medical conditions in babies or adults who do not yet show signs of impairment. The large quantities of DNA formed through PCR can be introduced into the genome of another organism to create a transgenic organism . Transgenic animals are important for creating animal models of human disease. For example, hereditary diseases such as Alzheimer's that do not normally occur in mice can be introduced into the mouse genome. When the mouse begins to show symptoms of the disease, scientists can administer different treatments to find out which is the most effective.

Transgenic plants can also be used instead of the application of toxic pesticides ; the goal is to create a plant that can defend itself against insects by producing its own insecticides that are not harmful to humans. Then fewer chemical pesticides would be needed, reducing the contamination of drinking water and harm to humans exposed to the chemicals.

Mark R. Hughes, deputy director of the National Center for Human Genome Research at the National Institutes of Health, the American base for the Human Genome Project , called PCR "the most important new scientific technology to come along in the last hundred years." (Powledge 1998). Its principle limitation is that the primer sequence must be known so that primers can be synthesized prior to the first cycle of PCR. Furthermore, PCR is less accurate when used to replicate large gene sequences (greater than approximately 5,000 nucleotides long), which means that it is difficult to study complex proteins. Additionally, the procedure is expensive and currently too technically demanding to be carried out by nonprofessionals. These drawbacks are being addressed by developments that would fully automate PCR, or provide simpler and less expensive kits. These kits could be used, for example, by people who suspect they are developing cancer. Indeed, PCR is expanding beyond the world of research and will be increasingly available to people for direct independent analyses.

see also Genes; Genetic Engineering; Genetics.

Rebecca M. Steinberg

Bibliography

Frank-Kamenetskii, M. D. Unraveling DNA: The Most Important Molecule of Life. Reading, MA: Addison-Wesley, 1997.

McPherson, M. J., S. G. Moller, R. Beynon, and C. Howe. PCR (Basics: From Background to Bench). Oxford: BIOS; New York: Springer, 2000.

Nair, P. K. G., and K. Prabhaker Achar. A Textbook of Genetics and Evolution. New Delhi: Konark; New York: Distributed by Advent Books, 1990.

Sninsky, John J. PCR Applications: Protocols for Functional Genomics, Michael A. Innis and David H. Glefand, eds. San Diego, CA: Academic Press, 1999.

PCR (Polymerase Chain Reaction)

PCR, or polymerase chain reaction, is a biochemical technique that can generate millions of copies of a template strand of DNA . The technique relies on the same enzymes that cells use to replicate DNA, however it is performed in a simple test tube using controlled cycles of heating and cooling. PCR has revolutionized the field of biotechnology, making it quick and inexpensive to replicate, or amplify, specific segments of DNA.

PCR was conceptualized by molecular biologist Kary Mullis in 1983. While driving the highway between San Francisco and Mendocino, California, Mullis realized that very simple molecules could be used to replicate DNA in vitro, given the proper conditions. Prior to PCR, molecular biologists relied on bacteria to make copies of DNA. This process was both slow and subject to inaccuracies. After developing a conceptual model for PCR, Mullis refined the technique over the next seven years while working for Cetus Corporation in Emoryville, California. In 1993, Mullis was awarded half of the Nobel Prize in Chemistry for his work.

The DNA molecule is a double helix, which means that it consists of two long strands of smaller molecules. These long strands twist around each other. Each strand is made up of a sequence of four different smaller molecules called nucleotides. The four nucleotides are adenine (A), guanine (G), cytosine (C), and thymine (T). Each nucleotide always associates itself with a complementary nucleotide so that if adenine is on one of the strands, thymine is found across from it on the other strand. Similarly, if cytosine is on one strand, guanine is found across from it on the other strand.

Each strand of DNA has an orientation. One end of the molecule is known as the 5′ (or 5 prime) end and the other is called the 3′ (or 3 prime) end. This is because each nucleotide contains a 5′-phosphate on one side and 3′hydroxyl on the other side. The nucleotides are linked together by a reaction between the phosphate and the hydroxyl. The nucleotide on one end of the strand has an unconnected phosphate, the 5′ end, and the nucleotide on the other end has an unconnected hydroxyl, the 3′ end. The two strands of DNA are oriented in opposite directions so that the 5′ end of one strand matches the 3′ end of the other.

In order to make copies of DNA, the two strands are first separated from each other. Then a short molecule called a primer attaches itself to a location toward the 5′ end of the part of the DNA to be replicated on one of the strands. A primer is usually about 20 nucleotides long. Next, a special enzyme called DNA polymerase attaches itself to primer. This enzyme has the unique ability to add nucleotides to a growing DNA molecule. DNA polymerase uses the original strand of DNA as a template as it, in effect, slides along the original strand of DNA and pieces together a strand of complementary nucleotides. If, for example, the original strand contains the sequence CGGTA, then the DNA polymerase builds a strand with a sequence GCCAT. Because of the complementary nature of the nucleotides that make up DNA, after the original strands are separated and copied by DNA polymerase, the result is two copies identical to the double-stranded original. DNA polymerase moves along the DNA in the 5′ to the 3′ direction only.

The primer is extremely important to DNA replication because DNA polymerase can only add nucleotides to a growing chain, it cannot begin a new molecule. In cells, the primer is often a piece of RNA that binds to the DNA on the 5′ end of a gene . In biotechnological applications, primers are synthesized so that specific portions of DNA are reproduced. In order to copy both strands of DNA for a specific gene, two primers are needed, one for each strand. These two primers are not simple complements of each other because, due to the orientation of the two strands, the two primers will attach to DNA on opposite sides of the gene.

The biochemicals required for PCR are: at least one strand of the target DNA; two primers, one for each strand of the DNA; the enzyme DNA polymerase; and the four nucleotides found in DNA, adenine, guanine, cytosine, and thymine. These molecules are all combined in an instrument that carefully controls the heat of the mixture.

The steps required for PCR are fundamentally simple. First the strands of DNA are separated from each other by heating them to about 90°C (194°F) for roughly 30 seconds. At this high temperature, DNA is denatured and does not form a double strand. As a result, the primers are unable to bind to the target DNA. In the second step, the mixture is cooled to about 55°C (131°F), a temperature at which the DNA molecule takes on its double-stranded conformation. During this step, the primers bind to each of the target DNA strands on the 5′ side of the region to be copied. An excess of primer is added to the mixture to ensure that the primers anneal to the target DNA strands rather than the target DNA strands reattaching to each other. This second step takes about 20 seconds. Finally, the temperature is raised to about 75°C (167°F), which is the temperature that the DNA polymerase most commonly used in PCR is most effective. The DNA polymerase then extends the complementary strand of DNA, which takes about a minute. The result, after the first cycle, is two complete copies of the target DNA.

The cycle is then repeated multiple times. The second time it is repeated, both the original target DNA and the newly synthesized strands are copied; the result is four complete copies of the target DNA. The third time the cycle is repeated, eight copies result and so on. Usually between 20 and 30 cycles are completed, taking just a few hours, and the result is between one million and one billion copies of the original target piece of DNA.

The DNA polymerase usually used in PCR is known as Taq polymerase, because it is derived from the bacterium Thermus aquaticus. This bacterium is thermophyllic, meaning that it lives in locations with very high ambient temperatures, such as hot springs. In particular, the DNA polymerase of T. aquaticus is thermally stable at temperatures as high as 95°C (203°F), and so the high heating required to separate the double strands of DNA has no effect on the molecule. In addition, at higher temperatures, the chance of a primer binding to non-target DNA decreases. Because the Taq polymerase operates optimally at 72°C (161°F), the specificity of the PCR reaction is high and the DNA copied by the process is homogeneous.

Because PCR can be used to generate a large number of copies of very small amounts of DNA in very little time, it has quickly become an extremely useful and popular technology. Only ten years after it was developed, PCR had been referenced in more than 7,000 scientific publications. The applications of PCR are so great that it has become a standard research tool.

In forensics, the field of DNA fingerprinting relies on PCR. A very small sample of blood , semen , hair root, or tissue can be used to identify a person using PCR on the DNA from the nucleus of cells. The Federal Bureau of Investigation houses a genetic database called CODIS (Combined DNA Index System) that holds genetic information on convicted criminals and missing persons. A sensitive technique that can be used to establish maternal relationships between people is called mitochondrial DNA analysis , which relies on PCR. Biological material that is degraded or very old or tissues that do not contain nuclei, such as hair shafts and bones, are often more likely to yield information using this technique instead of DNA fingerprinting.

PCR is also important in answering basic scientific questions. In the field of evolutionary biology, PCR has been used to establish relationships among species. In anthropology, it has used to understand ancient human migration patterns . In archaeology , it has been used to help identify ancient human remains. Paleontologists have used PCR to amplify DNA from extinct insects preserved in amber for 20 million years. The Human Genome Project, which had a goal of determining the sequence of the 3 billion base pairs in the human genome, relied heavily on PCR. The genes responsible for a variety of human diseases have been identified using PCR. For example, a PCR technique called multiplex PCR identifies a mutation in a gene in boys suffering from Duchenne muscular dystrophy. PCR can also be used to search for DNA from foreign organisms such as viruses or bacteria. For instance, the presence of the HIV virus that causes AIDS can be determined using PCR on blood cells.

see also DNA banks for endangered animals; DNA databanks; DNA fingerprint; DNA sequences, unique; Electrophoresis; Hair analysis; Mitochondrial DNA typing; RFLP (restriction fragment length polymorphism); STR (short tandem repeat) analysis; Y chromosome analysis.

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 100 times in a few hours. In other words, it allows for the replication of a specific strand of deoxyribonucleic acid (DNA) and was a remarkable boost to the fields of genetic engineering and biomedical research. American molecular biologist Kary Mullis (1944–) developed the techniques of PCR in the 1970s. For his ingenious work, he was awarded the 1993 Nobel Prize in chemistry.

PCR amplification of DNA is like any DNA replication by DNA polymerase in vivo ; that is, in living cells. The difference is that PCR produces DNA in a test tube.

For a PCR reaction to proceed, four components are necessary: template (a sequence of DNA), primer (a region of DNA at which the reaction begins), deoxyribonucleotides (adenine, thymine, cytosine, guanine; the four buidling blocks of DNA) and DNA polymerase (the enzyme that participates in the construction of new DNA). 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) to cause one strand of DNA to separate from the other strand in the DNA double helix. This separation is called denaturation. Each separated strand is capable of being a template. The second step of the PCR is carried out around 122°F(50°C). At this lowered temperature, the two primers join back together (anneal); the annealing is specific, with complimentary nucleotide sequences on the two strands linking with one another. The DNA polymerase then extends the primer using the nucleotides that are added in the reaction mixture. As a result, at the end of each cycle, the numbers of DNA molecules double. Since a cycle can be done in minutes, the amoutn of DNA can increase tremendously in a short time (hours), generating quantities of the target sequence that are suitable for study or for some other purpose.

PCR was initially carried out manually in incubators of different temperatures for each step until the extraction 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 95°C for many hours. Several additional heat-resistant DNA polymerases have also now been identified.

Genetically 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 thus workable—amount in a very short time. Researchers in the Human Genome Project made extensive use of PCR to look for markers in cloned DNA segments and to establish the proper arrangement of DNA from the many fragment of DNA generated during the sequencing process. Molecular biologists use PCR to produce exact copies (clones) of DNA from a target sequence. 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 amplification of DNA in a short time (this is sometimes termed “real time”). This enables quantitative detection of DNA molecules that exist in minute amounts. PCR is also used widely in clinical tests. Today, routine to use PCR in the diagnosis of infectious diseases such acquired immunodeficiency syndrome (AIDS) and in a number of forensic tests.

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 100 times in a few hours. American molecular biologist Kary Mullis developed the techniques of PCR in the 1970s. For his ingenious invention, he was awarded the 1993 Nobel Prize in physiology or medicine.

PCR amplification of DNA is like any DNA replication by DNA polymerase in vivo. (in lving cells) The difference is that PCR produces DNA in a test tube. For a PCR reaction to proceed, 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° (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 initially carried out manually in incubators of different temperatures for each step until the extraction 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 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 thus workable—amount in a very short time. Researchers in the Human Genome Project use 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, routine to use PCR in the diagnosis of infectious diseases such AIDS and in a number of forensic tests.

See also DNA synthesis; DNA technology; Fluorescence in situ hybridization (FISH); Forensic science; Gene chips and microarrays; Gene splicing; Gene therapy; Genetic engineering; Genetic identification of microorganisms.

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