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

Patent · Tue Jan 01 00:00:00 EST 1991

OSTI ID:867894

Gray, Joe W ^[1]; Pinkel, Daniel ^[2]

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1. Livermore, CA
2. Walnut Creek, CA

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.

• Research Organization: Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
• DOE Contract Number: W-7405-ENG-48
• Assignee: Regents of University of California (Oakland, CA)
• Patent Number(s): US 5028525
• OSTI ID: 867894
• Country of Publication: United States
• Language: English

Related Subjects

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/