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. The 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. No Drawings
- Inventors:
- Issue Date:
- OSTI Identifier:
- 7172951
- Patent Number(s):
- 5028525
- Application Number:
- PPN: US 7-543463
- Assignee:
- University of California, Oakland, CA (United States)
- DOE Contract Number:
- W-7405-ENG-48
- Resource Type:
- Patent
- Resource Relation:
- Patent File Date: 26 Jun 1990
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; DNA; BIOASSAY; DNA HYBRIDIZATION; DNA POLYMERASES; SPECIFICITY; ENZYMES; HYBRIDIZATION; NUCLEIC ACIDS; NUCLEOTIDYLTRANSFERASES; ORGANIC COMPOUNDS; PHOSPHORUS-GROUP TRANSFERASES; POLYMERASES; PROTEINS; TRANSFERASES; 550200* - Biochemistry
Citation Formats
Gray, J W, and Pinkel, D. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ. United States: N. p., 1991.
Web.
Gray, J W, & Pinkel, D. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ. United States.
Gray, J W, and Pinkel, D. Tue .
"Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ". United States.
@article{osti_7172951,
title = {Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ},
author = {Gray, J W and Pinkel, D},
abstractNote = {A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The 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. No Drawings},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Jul 02 00:00:00 EDT 1991},
month = {Tue Jul 02 00:00:00 EDT 1991}
}