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PMID: 6310503 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid.

Nucleic acids research ·Vol. 11 ·No. 16 ·1983-08-25 ·Pages 5521-40

Guo LH, Yang RC, Wu R

Abstract

A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.

MeSH Terms
Base Sequence Cloning, Molecular DNA/genetics DNA Restriction Enzymes DNA-Directed DNA Polymerase/metabolism Genetic Engineering/methods Plasmids
Chemicals
DNA DNA-Directed DNA Polymerase DNA Restriction Enzymes
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Guo L H
Yang R C
Wu R
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25 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/11.16.5521
Published
1983-08-25
Pages
5521-40
Language
English
Region
England
NLM ID
0411011
PMCID
PMC326295
Subset
IM
Grants
NIGMS NIH HHS · GM29179 · United States
External Links
PubMed source DOI: 10.1093/nar/11.16.5521 PMC Full Text View journal details
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