Method for producing labeled single-stranded nucleic acid probes

Patent · Tue Oct 19 00:00:00 EDT 1999

OSTI ID:872595

Dunn, John J ^[1]; Quesada, Mark A ^[2]; Randesi, Matthew ^[3]

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1. Bellport, NY
2. Middle Island, NY
3. Upton, NY

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

• Research Organization: Associated Universities, Inc., Upton, NY (United States)
• DOE Contract Number: AC02-76CH00016
• Assignee: Brookhaven Science Associates (Upton, NY)
• Patent Number(s): US 5968786
• Application Number: 09/215,817
• OSTI ID: 872595
• Country of Publication: United States
• Language: English

Related Subjects

method
producing
labeled
single-stranded
nucleic
acid
probes
disclosed
introduction
unidirectional
deletions
cloned
dna
segment
specifically
comprises
providing
recombinant
construct
comprising
inserted
cloning
vector
f1
endonuclease
recognition
sequence
adjacent
insertion
site
contacted
protein
pii
encoded
ii
phage
generating
nick
nicked
coli
exonuclease
iii
expanding
gap
gapped
single-strand-specific
linearized
molecule
containing
double-stranded
deletion
corresponding
size
treated
manner
incubated
ligase
conditions
appropriate
ligation
embodiment
produced
described
polymerase
presence
nucleotides
fill
digestion
restriction
enzyme
cuts
outside
product
denatured
produce
probe
stranded dna
dna probes
molecule containing
comprises providing
acid probes
dna polymerase
method comprises
nucleic acid
recombinant dna
dna segment
single-stranded nucleic
cloning vector
acid probe
cloned dna
dna molecule
single-stranded dna
restriction enzyme
recognition sequence
sequence adjacent
producing single-stranded
method comprise
unidirectional deletions
dna construct
f1 endonuclease
dna probe
dna ligase
endonuclease recognition
construct comprising
labeled single-stranded
labeled nucleotides
insertion site
nuclease recognition
stranded nucleic
producing single
/435/