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Title: Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

Abstract

A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

Inventors:
 [1];  [2]
  1. (Livermore, CA)
  2. (Walnut Creek, CA)
Issue Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
OSTI Identifier:
867894
Patent Number(s):
5028525
Assignee:
Regents of University of California (Oakland, CA) LLNL
DOE Contract Number:  
W-7405-ENG-48
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
method; preparing; applying; single; stranded; dna; probes; double; target; dnas; situ; provided; producing; non-self-complementary; nucleic; acid; treating; therewith; probe; constructed; restriction; enzyme; exonuclease; form; template; primers; polymerase; digested; strand; resynthesized; presence; labeled; nucleoside; triphosphate; precursor; fragments; separated; treated; construct; application; significantly; increases; efficiency; specificity; hybridization; mixtures; increasing; effective; concentration; eliminating; self-hybridization; reducing; amount; available; mismatched; hybridizations; significantly increases; single stranded; stranded dna; dna probes; acid probes; dna polymerase; nucleic acid; acid probe; target dna; restriction enzyme; double stranded; complementary nucleic; dna probe; significantly increase; nucleoside triphosphate; single strand; producing single; /435/436/

Citation Formats

Gray, Joe W., and Pinkel, Daniel. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ. United States: N. p., 1991. Web.
Gray, Joe W., & Pinkel, Daniel. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ. United States.
Gray, Joe W., and Pinkel, Daniel. Tue . "Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ". United States. https://www.osti.gov/servlets/purl/867894.
@article{osti_867894,
title = {Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ},
author = {Gray, Joe W. and Pinkel, Daniel},
abstractNote = {A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1991},
month = {1}
}

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