Using the Polymerase Chain Reaction to Amplify DNA

A major problem in working at the molecular level is that each gene is a tiny fraction of the total cellular DNA. Because each gene is rare, it must be isolated and amplified before it can be studied. Before mid-1980, the only procedure available for amplifying DNA was gene cloning—placing the gene in a bacterial cell and multiplying the bacteria. Cloning is labor intensive and requires at least several days to grow the bacteria. In 1983, Kary Mullis of the Cetus Corporation conceptualized a new technique for amplifying DNA in a test tube. The polymerase chain reaction allows DNA fragments to be amplified a billionfold within just a few hours. It can be used with extremely small amounts of original DNA, even a single molecule. The polymerase chain reaction has revolutionized molecular biology and is now one of the most widely used of all molecular techniques.

The basis of PCR is replication catalyzed by a DNA polymerase enzyme, which has two essential requirements:

(1) a single-stranded DNA template from which a new DNA strand can be copied and (2) a primer with a 3'-OH group to which new nucleotides can be added.

Because a DNA molecule consists of two nucleotide strands, each of which can serve as a template to produce a new molecule of DNA, the amount of DNA doubles with each replication event. The starting point of DNA synthesis on the template is determined by the choice of primers. The primers used in PCR are short fragments of DNA, typically from 17 to 25 nucleotides long, that are complementary to known sequences on the template. A different primer is used for each strand.

To carry out PCR, one begins with a solution that includes the target DNA (the DNA to be amplified), DNA polymerase, all four deoxyribonucleoside triphosphates (dNTPs—the substrates for DNA polymerase), primers that are complementary to short sequences on each strand of the target DNA, and magnesium ions and other salts that are necessary for the reaction to proceed. A typical poly-merase chain reaction includes three steps (I Figure 18.18).

ffi DNA is heated to 90°-100°C to separate the two strands.

^ The DNA is quickly cooled to 30°-65°C to allow short single-strand primers to anneal to their complementary sequences.

^ The solution is heated to 60°-70°C; DNA polymerase synthesizes new DNA strands,.

| .creating two new, double-stranded DNA molecules.

H The entire cyclel^^ is repeated.

| Each time the cycle is repeated, the amount of target DNA doubles.

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