Polymerase Chain Reaction (PCR) (World of Microbiology and Immunology)
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
PCR (Polymerase Chain Reaction) (World of Forensic Science)
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 5phosphate on one side and 3ydroxyl 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 5end, and the nucleotide on the other end has an unconnected hydroxyl, the 3end. The two strands of DNA are oriented in opposite directions so that the 5end of one strand matches the 3end 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 5end 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 5to the 3direction 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 5end 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 5side 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.