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PMID: 3474623 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 84 ·No. 14 ·1987-07-00 ·Pages 4767-71

Tabor S, Richardson CC

Abstract

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.

MeSH Terms
Base Sequence DNA, Recombinant/analysis DNA, Viral/analysis DNA-Directed DNA Polymerase/metabolism Deoxyribonucleotides/metabolism Exodeoxyribonucleases Free Radicals Nucleotide Mapping/methods Oxidation-Reduction Protein Multimerization T-Phages/enzymology Thioredoxins/metabolism Viral Proteins/metabolism
Chemicals
DNA, Recombinant DNA, Viral Deoxyribonucleotides Free Radicals Viral Proteins Thioredoxins bacteriophage T7 induced DNA polymerase DNA-Directed DNA Polymerase Exodeoxyribonucleases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Tabor S
Richardson C C
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19 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.84.14.4767
Published
1987-07-00
Pages
4767-71
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC305186
Subset
IM
Grants
NIAID NIH HHS · AI06045 · United States
External Links
PubMed source DOI: 10.1073/pnas.84.14.4767 PMC Full Text View journal details
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