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Protocols in Human Molecular Genetics highlights the tremendous advances in our ability to work on the human genome that have emerged in the past few years. The latest techniques are set forth in the clear, concise, easy-to-follow format that is the hallmark of Humana's Methods in Molecular Biology series. Nearly two-thirds of the book is devoted to describing practical procedures comprising the widest range of new methodologies in human molecular genetics, with the rest focusing on their specific experimental and clinical applications. An essential tool for everyone-whether novice or seasoned expert-involved in the rapidly growing area of human genome studies.
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CHAPTER1 The Polymerase
Chain Reaction
Getting Started
Charles
R. M. Bangham
1. Introduction The polymerase chain reaction (PCR) uses two oligonucleotide primers to direct the synthesis of specific sequences of DNA. One primer anneals to the coding strand of DNA and the other to the anticoding strand; the primer binding sites are typically separated by a few hundred base pairs (loo1000 bp). Repeated cycles of polymerization and denaturation lead to the exponential increase of the sequence defined by the primers. The extraordinary sensitivity and specihcity of PCR have established it as a standard technique in molecular biology in the short time since it was first described (1). The purpose of this chapter is to suggest starting conditions for a PCR reaction and ways to overcome the main problems in PCR. It is intended as a practical guide, so theoretical aspects will not be discussed in detail. For a fuller account, there are excellent and comprehensive guides edited by Erlich (2) and by Innis et al. (3‘). Protocols for special applications of PCR are described in later chapters in this volume.
2. Choice of Primers The ideal oligonucleotide l