POLYMERASE CHAIN REACTION (PCR) ASSAY
Although the DNA analysis systems described earlier are still used in research and clinical laboratories, a major revolution in detecting DNA material occured with the introduction of the PCR assay. This method is particularly valuable since it can markedly amplify a small piece of DNA before cleavage with a restriction enzyme. Complementary oligonucleotide primers from either end of the target DNA are added to the denaturated sample, along with a heat-resistant DNA polymerase. If the target sequence is present, the primers anneal to it and provide a starting point for the polymerase to begin the synthesis of second-strand DNA. The newly synthesized double-stranded DNA is then denaturated by heating and exposed again to the polymerase enzyme at a lower temperature. In this way, newly synthesized molecules and original DNA can reassociate with the primer and act as templates for further rounds of DNA synthesis. After completing about thirty cycles (usually two to three hours in an automated machine), the specific target sequence is amplified more than 1-million-fold. This powerful and sensitive technique can detect a specific DNA sequence from a single cell (eg lymphocyte, sperm), fixed pathological specimens, and dried blood spots. The main disadvantage is that contamination of the reaction mixture with traces of DNA from another source will lead to false positive results. Thus extreme care in handling specimens to be tested as well as in the test itself is obligatory.