Method for introducing unidirectional nested deletions

Details Method for introducing unidirectional nested deletions Method for introducing unidirectional nested deletions

1997-11-10

1999-07-27

McKelvey, Terry

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C12N 1564

Patent

active

059289082

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. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

REFERENCES:
patent: 4843003 (1989-06-01), Henikoff et al.
patent: 4889799 (1989-12-01), Henikoff et al.
patent: 5354656 (1994-10-01), Sorge et al.
Greenstein et al, The Journal of Biological Chemistry, vol. 264 (21): pp. 12627-12632, 1989.
Chang et al., Gene 127: 95-98 (1993).
Life Technologies, GIBCO BRL Products & Reference Guide: pp. 19-14, 19-15, and 19-32 (1997/1998).

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