Principles of the Polymerase Chain Reaction

Abstract

The polymerase chain reaction (PCR) is a nucleotide sequence amplification procedure allowing the production of large amounts of a specific DNA or RNA sequence from a complex DNA or RNA template. It is based on oligonucleotide primer annealing onto complementary nucleic acid sequences followed by enzymatic DNA synthesis by application of a heat-stable DNA polymerase. These steps, together with melting of the double-stranded DNA to obtain single-stranded templates, are performed sequentially using a computerized thermocycler which allows the automatic control of melting, annealing, and synthesis temperature during some 25-35 cycles. From the starting amount of as low as one single DNA or RNA molecule, several hundred thousand or even millions of copies of the initial sequence may be obtained. During the last decade, the technique has been widely applied for in vitro studies, and, very recently, in situ PCR techniques have been developed. The following chapter will familiarize the readers with the basic principles of this exciting technique. As an example of application, our results on the diagnosis of cystic fibrosis are presented.