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

Patent · 02 July 1991

OSTI ID:7172951

Gray, J W; Pinkel, D

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

• DOE Contract Number: W-7405-ENG-48
• Assignee: University of California, Oakland, CA (United States)
• Patent Number(s): US 5028525; A
• Application Number: PPN: US 7-543463
• OSTI ID: 7172951
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BIOASSAY
DNA
DNA HYBRIDIZATION
DNA POLYMERASES
ENZYMES
HYBRIDIZATION
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTEINS
SPECIFICITY
TRANSFERASES