Principle of PCR for Laboratory Diagnosis of Disease
PCR is based on the enzymatic amplification of a fragment of DNA that is flanked by two 'primers', short oligonucleotides that hybridize to the opposite strands of the target sequence and then prime synthesis of the complementary DNA sequence by DNA polymerase (an enzyme). The chain reaction is a three-step process, denaturation, annealing, and extension, that is repeated in several cycles. At each stage of the process, the number of copies is doubled from two to four, to eight, and so on. The reactions are controlled by changing the temperature using a special heat-stable Taq polymerase. After 20 cycles, roughly 1 million copies exist, or enough material to detect the desired DNA by conventional means such as color reaction.
RNA can also be studied by making a DNA copy of the RNA using the enzyme reverse transcriptase. Such an approach enables the study of mRNA in cells that use the molecule to synthesize specific proteins or the detection of the genome of RNA viruses. PCR has been fully automated via use of thermal cycling. It is a fast, sensitive, and specific test with applications in diagnosis of various diseases described in following chapters.