Polymerase Chain Reaction ( PCR )

Abstract

Abstract Polymerase chain reaction (PCR) is a rapid, in vitro deoxyribonucleic acid (DNA) synthesis process, which can amplify up to a billion copies of a given nucleic acid target. It has been extensively applied for the identification, detection and diagnosis of genetic and infectious disease. In the article, we provide a general overview of the PCR process, describe methods to identify and minimize exogenous contamination, and highlight limitations of product size and polymerase‐dependent errors. We present many of the applications for which PCR is used, particularly in creating recombinant DNA constructs, in detecting genetic variation, in preparing templates for DNA sequence analysis and in quantitating gene transcripts or viral copies. Whole genome amplification and the ligation chain reaction are also presented, which we believe this article provides the reader with a comprehensive viewpoint of the variety of amplification‐based methods used in molecular biology today. Key Concepts Polymerase chain reaction is an in vitro amplification method widely used in molecular biology. PCR contamination can occur from a single molecule of foreign or exogenous DNA, which, if gone unchecked, can confound the interpretation of results. PCR cloning of full‐length genes can be problematic because mutations can result in amino acid substitutions in wild‐type sequences. PCR can reliably amplify target sizes up to 3–4 kb from a variety of source materials. Recombinant PCR products can be created by mismatching sequences between the primer and the template DNA or by adding 5′ exogenous sequences to the primers. PCR can detect human genetic variation associated with hereditary disease and cancer. Degenerate PCR is a powerful tool for identifying novel gene family members that are important in drug development. Quantitative PCR has been widely used for studying gene expression and estimating viral copy number. Emulsion PCR is used to prepare templates in a cell‐free system for next‐generation sequencing platforms. Whole genome amplification uses the highly processive φ29 DNA polymerase to amplify nanogram quantities of precious samples into microgram amounts.