Method for introducing unidirectional nested deletions
Abstract
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.more »
- Inventors:
-
- Bellport, NY
- Middle Island, NY
- Upton, NY
- Issue Date:
- Research Org.:
- Associated Universities, Inc., Upton, NY (United States)
- OSTI Identifier:
- 872405
- Patent Number(s):
- 5928908
- Application Number:
- 08/966,958
- Assignee:
- Brookhaven Science Associates (Upton, NY)
- Patent Classifications (CPCs):
-
C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES
- DOE Contract Number:
- AC02-76CH00016
- Resource Type:
- Patent
- Country of Publication:
- United States
- Language:
- English
- Subject:
- method; introducing; unidirectional; nested; deletions; disclosed; introduction; 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; single-stranded; nick; nicked; coli; exonuclease; iii; expanding; gap; gapped; single-strand-specific; producing; linearized; molecule; containing; double-stranded; deletion; corresponding; size; treated; manner; incubated; ligase; conditions; appropriate; ligation; probes; embodiment; produced; described; polymerase; presence; labeled; nucleotides; fill; digestion; restriction; enzyme; cuts; outside; product; denatured; produce; nucleic; acid; probe; stranded dna; dna probes; molecule containing; comprises providing; 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; nested deletions; method comprise; unidirectional deletions; dna construct; f1 endonuclease; dna probe; dna ligase; endonuclease recognition; construct comprising; introducing unidirectional; labeled single-stranded; labeled nucleotides; unidirectional nested; insertion site; nuclease recognition; stranded nucleic; producing single; /435/
Citation Formats
Dunn, John J, Quesada, Mark A, and Randesi, Matthew. Method for introducing unidirectional nested deletions. United States: N. p., 1999.
Web.
Dunn, John J, Quesada, Mark A, & Randesi, Matthew. Method for introducing unidirectional nested deletions. United States.
Dunn, John J, Quesada, Mark A, and Randesi, Matthew. Tue .
"Method for introducing unidirectional nested deletions". United States. https://www.osti.gov/servlets/purl/872405.
@article{osti_872405,
title = {Method for introducing unidirectional nested deletions},
author = {Dunn, John J and Quesada, Mark A and Randesi, Matthew},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Jul 27 00:00:00 EDT 1999},
month = {Tue Jul 27 00:00:00 EDT 1999}
}
Works referenced in this record:
Double-strand Cleavage and Strand Joining by the Replication Initiator Protein of Filamentous Phage f1
journal, July 1989
- Greenstein, D.; Horiuchi, K.
- Journal of Biological Chemistry, Vol. 264, Issue 21
The Exo-gap method employing the phage f1 endonuclease generates a nested set of unidirectional deletions
journal, May 1993
- Chang, David W.; Tartof, Kenneth D.; Yeung, Anthony T.
- Gene, Vol. 127, Issue 1