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PMID: 347445 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 75 ·No. 4 ·1978-04-00 ·Pages 1763-7

Goff SP, Berg P

Abstract

A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.

MeSH Terms
DNA, Bacterial DNA, Circular DNA, Recombinant Escherichia coli Exonucleases/metabolism Plasmids Poly dA-dT Polydeoxyribonucleotides
Chemicals
DNA, Bacterial DNA, Circular DNA, Recombinant Polydeoxyribonucleotides Poly dA-dT Exonucleases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Goff S P
Berg P
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23 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.75.4.1763
Published
1978-04-00
Pages
1763-7
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC392420
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.75.4.1763 PMC Full Text View journal details
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