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An unprecedented collection of core PCR techniques useful for the study and diagnosis of human diseases. Cutting-edge and essential for today's diagnostic laboratories, these techniques heavily utilize nonisotopic, solution phase, and in situ amplification methods. A significant number of chapters describe applications exploiting the eggwhite sensitivity of PCR in the detection of rare or single cells, as in identifying fetal cells circulating in maternal blood, preimplantation embryo diagnosis, or finding circulating cancer cells. The book demonstrates over and over again the power of PCR-its high sensitivity, specificity, and ability to rapidly discriminate sequence variations.
E-Book Content
1 Introduction to the Polymerase Chain Reaction Y. M. Dennis Lo 1. Introduction The polymerase chain reaction (PCR) is an in vitro method for the amplification of DNA that was mtroduced in 1985 (I). The principle of the PCR is elegantly simple but the resulting method is extremely powerful. The adoption of the thermostable Taq polymerase in 1988 greatly simplifies the process and enables the automatron of PCR (2). Since then a large number of apphcatrons have been developed that are based on the basic PCR theme. The versatility and speed of PCR have revolutionized molecular diagnostics, allowmg the realization of a number of applications that were impossible in the pre-PCR era. This chapter offers an mtroductory guide to the process. 2. Principle of the PCR PCR may be regarded as a simplified version of the DNA rephcation processthat occurs during cell division. Basic PCR consrstsof three steps: thermal denaturation of the target DNA, primer annealing of synthetic oligonucleotide primers, and extension of the annealed primers by a DNA polymerase (Fig. 1). This three step cycle is then repeated a number of times, each time approximately doubling the number of product molecules. The am