Effects of Different DNA Polymerases in Ligation-Mediated PCR: Enhanced Genomic Sequencing and In vivo Footprinting
- Creators
- Garrity, Paul A.
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Wold, Barbara J.
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.
Additional Information
© 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.Attached Files
Published - 1021.pdf
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Additional details
- PMCID
- PMC48377
- Eprint ID
- 700
- Resolver ID
- CaltechAUTHORS:GARpnas92
- Muscular Dystrophy Association
- NIH Predoctoral Fellowship
- H600021
- Created
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2005-09-15Created from EPrint's datestamp field
- Updated
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2019-10-02Created from EPrint's last_modified field