12. PCR

Amplification of DNA by the Polymerase Chain Reaction

Molecular cloning allows individual DNA fragments to be propagated in bacteria and isolated in large amounts. An alternative method to isolating large amounts of a single DNA molecule is the polymerase chain reaction (PCR), which was developed by Kary Mullis in 1988. Provided that some sequence of the DNA molecule is known, PCR can achieve a striking amplification of DNA via reactions carried out entirely in vitro. Essentially, DNA polymerase is used for repeated replication of a defined segment of DNA. The number of DNA molecules increases exponentially, doubling with each round of replication, so a substantial quantity of DNA can be obtained from a small number of initial template copies. For example, a single DNA molecule amplified through 30 cycles of replication would theoretically yield 230 (approximately 1 billion) progeny molecules. Single DNA molecules can thus be amplified to yield readily detectable quantities of DNA that can be isolated by molecular cloning or further analyzed directly by restriction endonuclease digestion or nucleotide sequencing.

The general procedure for PCR amplification of DNA is illustrated in Figure 3.27. The starting material can be either a cloned DNA fragment or a mixture of DNA molecules-for example, total DNA from human cells. A specific region of DNA can be amplified from such a mixture, provided that the nucleotide sequence surrounding the region is known so that primers can be designed to initiate DNA synthesis at the desired point. Such primers are usually chemically synthesized oligonucleotides containing 15 to 20 bases of DNA. Two primers are used to initiate DNA synthesis in opposite directions from complementary DNA strands. The reaction is started by heating the template DNA to a high temperature (e.g., 95°C) so that the two strands separate. The temperature is then lowered to allow the primers to pair with their complementary sequences on the template strands. DNA polymerase then uses the primers to synthesize a new strand complementary to each template. Thus in one cycle of amplification, two new DNA molecules are synthesized from one template molecule. The process can be repeated multiple times, with a twofold increase in DNA molecules resulting from each round of replication.

The multiple cycles of heating and cooling involved in PCR are performed by programmable heating blocks called thermocyclers. The DNA polymerases used in these reactions are heat-stable enzymes from bacteria such as Thermus aquaticus, which lives in hot springs at temperatures of about 75°C. These polymerases are stable even at the high temperatures used to separate the strands of double-stranded DNA, so PCR amplification can be performed rapidly and automatically. RNA sequences can also be amplified by this method if reverse transcriptase is used to synthesize a cDNA copy prior to PCR amplification.

If enough of the sequence of a gene is known that primers can be specified, PCR amplification provides an extremely powerful method of obtaining readily detectable and manipulable amounts of DNA from starting material that may contain only a few molecules of the desired DNA sequence in a complex mixture of other molecules. For example, defined DNA sequences of up to several kilobases can be readily amplified from total genomic DNA, or a single cDNA can be amplified from total cell RNA. These amplified DNA segments can then be further manipulated or analyzed, for example, to detect mutations within a gene of interest. PCR is thus a powerful addition to the repertoire of recombinant DNA techniques. Its power is particularly apparent in applications such as the diagnosis of inherited diseases, studies of gene expression during development, and forensic medicine.

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