Chapter 4 - PCR Amplification: Capabilities and Cautions

Abstract

Forensic science and DNA-typing laboratories have greatly benefited from the discovery of a technique known as the polymerase chain reaction or PCR. PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence. This molecular “Xeroxing” process involves heating and cooling samples in a precise thermal cycling pattern over ≈30 cycles. The PCR amplification process has a dual purpose: to increase the number of molecules representing a specific target site, and to attach a label, most often a fluorescent dye, that enables detection of the amplicons produced. Both the amplification and labeling elements of the PCR process enhance detection sensitivity and specificity. In addition, a wide range of PCR cycling protocols have been used for various molecular biology applications. The primary reason that PCR protocols vary is that different primer sequences have different hybridization properties and thus anneal to the DNA template strands at different rates. Annealing time and temperature are some of the most critical parameters to optimize with multiplex PCR.