Method for introducing unidirectional nested deletions
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.
- Sponsoring Organization:
- USDOE; USDOE, Washington, DC (United States)
- DOE Contract Number:
- AC02-76CH00016
- Assignee:
- Brookhaven Science Associates, Upton, NY (United States)
- Patent Number(s):
- US 5928908; A
- Application Number:
- PPN: US 8-966958
- OSTI ID:
- 6376292
- Resource Relation:
- Patent File Date: 10 Nov 1997
- Country of Publication:
- United States
- Language:
- English
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Method for producing labeled single-stranded nucleic acid probes
Method for producing labeled single-stranded nucleic acid probes
Related Subjects
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