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

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
Authors:
;
Publication Date:
OSTI Identifier:
7172951
Report Number(s):
US 5028525; A
PPN: US 7-543463
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