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Effects of Different DNA Polymerases in Ligation-Mediated PCR: Enhanced Genomic Sequencing and In vivo Footprinting

Garrity, Paul A. and Wold, Barbara J. (1992) Effects of Different DNA Polymerases in Ligation-Mediated PCR: Enhanced Genomic Sequencing and In vivo Footprinting. Proceedings of the National Academy of Sciences of the United States of America, 89 (3). pp. 1021-1025. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:GARpnas92

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Abstract

We have developed a simplified procedure for the ligation-mediated polymerase chain reaction (LMPCR) using Thermococcus litoralis DNA polymerase (Vent DNA polymerase). We show that Vent DNA polymerase produces correct, blunt-ended primer extension products with substantially higher efficiency than Thermus aquaticus (Taq) DNA polymerase or modified T7 DNA polymerase (Sequenase). This difference leads to significantly improved genomic sequencing, methylation analysis, and in vivo footprinting with LMPCR. These improvements include representation of all bands with more uniform intensity, clear visualization of previously difficult regions of sequence, and reduction in the occurrence of spurious bands. It also simplifies the use of DNase I cut DNA for LMPCR footprinting.


Item Type:Article
Additional Information:Copyright © 1992 by National Academy of Sciences Communicated by Norman Davidson, October 8, 1991 (received for review September 4, 1991) We thank Paul Mueller for generous gifts of DNA, comments on the manuscript, and many helpful discussions; Linda Huang and Jeff Miner for advice and comments on the manuscript; and Joe Hacia and Pete Mathers for assistance and discussions. This work was supported by grants from the Muscular Dystrophy Association and National Institutes of Health to B.J.W. and by Predoctoral Training Award H600021. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:methylation, Vent DNA polymerase, terminal transferase, DNase I, transcription
Record Number:CaltechAUTHORS:GARpnas92
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:GARpnas92
Alternative URL:http://www.pnas.org/cgi/content/abstract/89/3/1021
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:700
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:15 Sep 2005
Last Modified:26 Dec 2012 08:41

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