PCR

  The concept of clonal amplification forms the basis for the polymerase chain reaction (PCR), a recently developed alternative to the cut-and-paste manipulations of bacterial cloning. PCR is an ingenious test-tube amplification procedure, based on the DNA replication machinery of the cell itself, which in many cases surpasses bacterial cloning in sensitivity, selectivity, and speed. The segment of DNA sequence to be amplified is combined with two short, chemically synthesized single-stranded DNA fragments, or oligonucleotide primers. The primers initiate the amplification, which proceeds in successive copying cycles, each of which doubles the number of DNA segments. A cycle begins with the melting of the double-stranded DNA starting material (called the template) into single strands. The separation of the DNA strands allows the two primers to anneal to their respective complementary sequence on the template, one at the end of each strand. A special heat-stable DNA polymerase enzyme begins adding nucleotide bases at the ends of the primers, extending the double-stranded helices that the primers have initiated on each strand of the template. By the time the polymerase has reached the end of the template, the desired double-stranded sequence has been reformed. The cycle then starts again, heating and separating the new double helices. Each cycle doubles the number of DNA templates (Figure 4, bottom). In theory, this process can repeat itself until it incorporate all the primers and free nucleotides into double-stranded DNA. It is possible to generate millions of copies of a DNA segment in a matter of hours with PCR, even when the starting material is only a single molecule of DNA. The two methods of gene cloning and amplification, PCR and bacterial cloning, have special applications and limitations. Nevertheless, the objectives of both are ultimately the same: to produce enough genetically identical material for biochemical analysis and further manipulation.

  The DNA sequence to be amplified is determined by the choice of primers, short synthetic oligonucleotides whose sequence corresponds to the flanking genomic DNA. After an excess of primers is mixed with the DNA to be amplified together with a heat-stable DNA polymerase, the strands of both the genomic DNA and the primers are separated by heating, and allowed to cool. Using the genomic DNA as a template, the polymerase elongates the primers binding to either strand, generating two new identical double-stranded DNA molecules, and doubling the number of DNA fragments. Each cycle is completed in a few minutes. Subsequent cycles of heating, cooling and polymerization of the new DNA strands produces a quick geometric amplification of the original DNA fragment.