Method for introducing unidirectional nested deletions

Patent · Tue Jul 27 04:00:00 EDT 1999

OSTI ID:872405

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

---

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: ASSOC UNIVERSITIES INC
• DOE Contract Number: AC02-76CH00016
• Assignee: Brookhaven Science Associates (Upton, NY)
• Patent Number(s): US 5928908
• Application Number: 08/966,958
• OSTI ID: 872405
• Country of Publication: United States
• Language: English

Related Subjects

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