Method for producing labeled single-stranded nucleic acid probes
US5968786A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Dec 18, 1998 |
| Grant date | Oct 19, 1999 |
| Priority date | — |
| Expiry date | Dec 18, 2018 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N15/102
- WIPO fieldBiotechnology
- WIPO sectorChemistry
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…
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.