Covalent joining of DNA to RNA by vaccinia topoisomerase and uses thereof
US7662556B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Sep 19, 2003 |
| Grant date | Feb 16, 2010 |
| Priority date | — |
| Expiry date | Apr 24, 2026 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Y599/01002
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The present invention provides a method of covalently joining a DNA strand to an RNA strand comprising (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5′ single-strand tails; and (b) adding to the topoisomerase-DNA intermediate an acceptor RNA strand complementary to the 5′ single-strand tail under conditions permitting a ligation of the covalently bound DNA strand of the topoisomerase-DNA intermediate to the RNA acceptor strand and dissociation of the topoisomerase, thereby covalently joining the DNA strand to the RNA strand. The present invention also provides a method of tagging a 5′ end of an RNA molecule. The present invention further provides a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase. The present invention also provides a method of tagging a 5′ end of an mRNA. The present invention provides a method of isolating and cloning full-length gene sequences using capped mRNA after subtraction of non-capped RNA.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.