Biochemistry II: The Polymerase Chain Reaction

The Polymerase Chain Reaction

You can isolate virtually any DNA sequence by means of the polymerase chain reaction, or PCR. PCR uses repetitive cycles of primer-dependent polymerization to amplify a given DNA. Very little original DNA is required, as long as two unique primers are available. Knowing the sequences of the primers before starting out is helpful, but not always necessary. Each cycle of PCR involves three steps: DNA double strand separation, primer hybridization, and copying. First, the original DNA is denatured by heat treatment to make two separated strands. Then the two primers are hybridized to the DNA, one to each of the two separated strands. These primers act as initiators for DNA polymerase, which copies each strand of the original double-stranded DNA. The original two strands of DNA now become four strands, which are then denatured. These four strands are then hybridized with the primers and each of them is now copied, to make eight strands, and so forth. Amplified DNA can be analyzed by any of the techniques used for analyzing DNA: it can be separated by electrophoresis, Southern blotted, or cloned. Because a single DNA sequence is obtained by PCR, sequence information can also be obtained directly. See Figure 1 .