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Title: 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 has 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 » The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe. 1 fig.« less

Inventors:
; ;
Issue Date:
Sponsoring Org.:
USDOE; USDOE, Washington, DC (United States)
OSTI Identifier:
6376292
Patent Number(s):
5928908 A
Application Number:
PPN: US 8-966958
Assignee:
Brookhaven Science Associates, Upton, NY (United States) PTO; EDB-99-086042
DOE Contract Number:  
AC02-76CH00016
Resource Type:
Patent
Resource Relation:
Patent File Date: 10 Nov 1997
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BIOTECHNOLOGY; DNA POLYMERASES; DNA SEQUENCING; ENDONUCLEASES; MOLECULAR BIOLOGY; RECOMBINANT DNA; DNA; DNA-ASE; ENZYMES; ESTERASES; HYDROLASES; NUCLEIC ACIDS; NUCLEOTIDYLTRANSFERASES; ORGANIC COMPOUNDS; PHOSPHODIESTERASES; PHOSPHORUS-GROUP TRANSFERASES; POLYMERASES; PROTEINS; STRUCTURAL CHEMICAL ANALYSIS; TRANSFERASES; 550200* - Biochemistry

Citation Formats

Dunn, J.J., Quesada, M.A., and Randesi, M. Method for introducing unidirectional nested deletions. United States: N. p., 1999. Web.
Dunn, J.J., Quesada, M.A., & Randesi, M. Method for introducing unidirectional nested deletions. United States.
Dunn, J.J., Quesada, M.A., and Randesi, M. Tue . "Method for introducing unidirectional nested deletions". United States.
@article{osti_6376292,
title = {Method for introducing unidirectional nested deletions},
author = {Dunn, J.J. and Quesada, M.A. and Randesi, M.},
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 has 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. 1 fig.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1999},
month = {7}
}