UNIT 15.3 Ligation-Mediated PCR for Genomic Sequencing and Footprinting
Published Online: 1 NOV 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 NOV 2001
- Published Print: OCT 2001
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.