Unit

UNIT 15.3 Ligation-Mediated PCR for Genomic Sequencing and Footprinting

  1. Paul R. Mueller1,
  2. Barbara Wold1,
  3. Paul A. Garrity2

Published Online: 1 NOV 2001

DOI: 10.1002/0471142727.mb1503s56

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Mueller, P. R., Wold, B. and Garrity, P. A. 2001. Ligation-Mediated PCR for Genomic Sequencing and Footprinting. Current Protocols in Molecular Biology. 56:15.3:15.3.1–15.3.26.

Author Information

  1. 1

    California Institute of Technology, Pasadena, California

  2. 2

    University of California, Los Angeles, Los Angeles, California

Publication History

  1. Published Online: 1 NOV 2001
  2. Published Print: OCT 2001

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.