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PMID: 3808957 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Reverse Southern hybridization.

Nucleic acids research ·Vol. 14 ·No. 24 ·1986-12-22 ·Pages 9943-54

Gamper HB, Cimino GD, Isaacs ST, Ferguson M, Hearst JE

Abstract

A DNA oligomer 25 nucleotides long which contained an HMT (4'-hydroxymethyl-4,5', 8-trimethylpsoralen) furan side monoadduct to thymidine at a 5'-TpA-3' site was used as a probe for the polylinker sequence present in single-stranded M13 mp19 DNA and in double-stranded pUC 19 DNA. Hybridization and photofixation were carried out simultaneously in solution under conditions approximating the melting temperature of the probe-target hybrid. Use of probe concentrations greater than 10(-8) M permitted hybridization times of a few minutes. Irradiation with near ultraviolet light converted the HMT monoadduct present in hybrid complexes into an interstrand crosslink. Efficient photofixation removed hybrid from the equilibrium distribution and resulted in the formation of additional probe-target complex. After removal of excess probe by centrifugation through a semi-permeable membrane (Centricon-30), samples were electrophoresed through an alkaline agarose gel which was analyzed by autoradiography. When using an HMT-modified 25-mer probe end-labeled with 3,000 Ci/mmole 32P, 0.015 ng (3.8 X 10(6) copies) of M13 DNA could be detected. With this same probe 10 micrograms of denatured human DNA (corresponding to 3.0 X 10(6) copies) did not give a signal.

MeSH Terms

DNA/genetics Kinetics Methods Nucleic Acid Denaturation Nucleic Acid Hybridization Oligodeoxyribonucleotides/chemical synthesis Photochemistry Plasmids

Chemicals

Oligodeoxyribonucleotides DNA

Authors & Affiliations

5 authors, click to expand affiliations / ORCID

Gamper H B

Cimino G D

Isaacs S T

Ferguson M

Hearst J E

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23 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/14.24.9943

Published

1986-12-22

Pages

9943-54

Language

English

Region

England

NLM ID

0411011

PMCID

PMC341346

Subset

External Links

PubMed source DOI: 10.1093/nar/14.24.9943 PMC Full Text View journal details

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