Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ
Patent
·
OSTI ID:867894
- Livermore, CA
- 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
- 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
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Related Subjects
/435/436/
acid
acid probe
acid probes
amount
application
applying
available
complementary nucleic
concentration
construct
constructed
digested
dna
dna polymerase
dna probe
dna probes
dnas
double
double stranded
effective
efficiency
eliminating
enzyme
exonuclease
form
fragments
hybridization
hybridizations
increases
increasing
labeled
method
mismatched
mixtures
non-self-complementary
nucleic
nucleic acid
nucleoside
nucleoside triphosphate
polymerase
precursor
preparing
presence
primers
probe
probes
producing
producing single
provided
reducing
restriction
restriction enzyme
resynthesized
self-hybridization
separated
significantly
significantly increase
significantly increases
single
single strand
single stranded
situ
specificity
strand
stranded
stranded dna
target
target dna
template
therewith
treated
treating
triphosphate
acid
acid probe
acid probes
amount
application
applying
available
complementary nucleic
concentration
construct
constructed
digested
dna
dna polymerase
dna probe
dna probes
dnas
double
double stranded
effective
efficiency
eliminating
enzyme
exonuclease
form
fragments
hybridization
hybridizations
increases
increasing
labeled
method
mismatched
mixtures
non-self-complementary
nucleic
nucleic acid
nucleoside
nucleoside triphosphate
polymerase
precursor
preparing
presence
primers
probe
probes
producing
producing single
provided
reducing
restriction
restriction enzyme
resynthesized
self-hybridization
separated
significantly
significantly increase
significantly increases
single
single strand
single stranded
situ
specificity
strand
stranded
stranded dna
target
target dna
template
therewith
treated
treating
triphosphate