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PMID: 1736283 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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 ·Vol. 89 ·No. 3 ·1992-02-01 ·Pages 1021-5

Garrity PA, Wold BJ

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.

MeSH Terms
Animals Bacteria/enzymology Base Sequence Binding Sites DNA/chemistry,ultrastructure DNA Nucleotidylexotransferase/metabolism DNA-Binding Proteins/metabolism DNA-Directed DNA Polymerase/metabolism Deoxyribonuclease I L Cells Methylation Mice Molecular Sequence Data Polymerase Chain Reaction/methods Sulfuric Acid Esters/chemistry Taq Polymerase Templates, Genetic
Chemicals
DNA-Binding Proteins Sulfuric Acid Esters DNA Taq Polymerase bacteriophage T7 induced DNA polymerase DNA Nucleotidylexotransferase DNA-Directed DNA Polymerase Deoxyribonuclease I dimethyl sulfate
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Garrity P A
Division of Biology, California Institute of Technology, Pasadena 91125.
Wold B J
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17 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.89.3.1021
Published
1992-02-01
Pages
1021-5
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC48377
Subset
IM
Grants
PHS HHS · H600021 · United States
External Links
PubMed source DOI: 10.1073/pnas.89.3.1021 PMC Full Text View journal details
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