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:
-
- Livermore, CA
- Walnut Creek, CA
- Issue Date:
- Research Org.:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- OSTI Identifier:
- 867894
- Patent Number(s):
- 5028525
- Assignee:
- Regents of University of California (Oakland, CA)
- Patent Classifications (CPCs):
-
C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
Y - NEW / CROSS SECTIONAL TECHNOLOGIES Y10 - TECHNICAL SUBJECTS COVERED BY FORMER USPC Y10T - TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- 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 = {Tue Jan 01 00:00:00 EST 1991},
month = {Tue Jan 01 00:00:00 EST 1991}
}