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Ligation‐Mediated PCR for Genomic Sequencing and Footprinting

2001; Wiley; Volume: 56; Issue: 1 Linguagem: Inglês

10.1002/0471142727.mb1503s56

1934-3639

Paul R. Mueller, B Wold, Paul Garrity,

RNA and protein synthesis mechanisms

Abstract This unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single‐sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation‐based addition of a unique DNA linker. This linker, together with the flanking gene‐specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete‐length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single‐base resolution. The ligation‐based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.