21] Use of polymerase chain reaction to amplify segments outside boundaries of known sequences

Abstract

This chapter reviews the procedures adopted by several investigators to obtain fragments outside a region of known sequence and provide a protocol for one technique––inverse polymerase chain reaction (PCR)––used by the laboratory. Prior to the implementation of the PCR, the acquisition of specific DNA fragments usually entailed the construction and screening of DNA libraries, and the traditional approach for walking from regions of known sequence into flanking DNA involved the successive probing of libraries with clones obtained from prior screenings. Nevertheless, several applications, including the recovery of the flanking sequence and the generation of end-specific hybridization probes for chromosome walking, can be greatly facilitated by the PCR. The specificity of the reaction is, in most cases, supplied by the unique primer designed to the core region. No single method can be recommended for every application because the source (DNA, RNA, or inferred from protein sequence) and size of the core region as well as the requirements for flanking information vary with each experiment.