Polymerase Chain Reaction: Principle, Technique and Applications in Pathology

Abstract

Polymerase chain reaction (PCR) is one of the most important techniques in molecular pathology by which the single or the pieces of target DNA are amplified by using a pair of DNA primer, heat-resistant DNA polymerase enzyme and nucleotides. This chapter discusses the principle, steps and application of PCR in pathology. There are four basic steps of PCR: denaturation, annealing and extension. The PCR thermal cycle rapidly heats and cools the PCR reagent mixture. The cycling time depends on (1) size of the DNA template and (2) G-C content of DNA. The number of the thermal cycler is usually set as 25--30 cycles. The PCR products are demonstrated by agarose gel electrophoresis of the product, cloning or sequencing of the products. The chapter also covers the troubleshooting of PCR. There are different types of PCR methods for diagnostic purposes that include reverse transcriptase PCR, asymmetric PCR, hot start PCR, in situ PCR, inverse PCR, single-strand conformation polymorphism, real-time PCR and nested PCR. All these types of PCR have been described in details.