Back to results
US 6,653,106 B1Granted
Topoisomerase-based ligation and cloning methods
AssigneeSloan-Kettering Institute for Cancer Research
InventorsStewart Shuman, JoAnn Sekiguchi
Life of this patent
Estimating the term…
Abstract
The present invention provides a method for covalently joining a DNA strand to an RNA strand using a topoisomerase enzyme. This invention also provides a method for obtaining a cDNA corresponding to a gene using a topoisomerase enzyme.
Claims17
A method for covalently joining a DNA strand and an RNA strand, the method comprising:
- (a) contacting a sequence-specific type I DNA topoisomerase in vitro with a double-stranded DNA whose first strand is to be covalently joined to the RNA strand, wherein the first strand comprises a recognition sequence for the type I topoisomerase at a site whose complement on the second strand is located 3′ of the 5′ terminus of the second strand, so as to permit the topoisomerase to bind to the DNA and cleave the first strand thereof, thereby forming a topoisomerase/DNA complex wherein the DNA's second strand has a 5′ tail;
- and
- (b) contacting the resulting complex in vitro with a 5′ OH-containing RNA strand whose 5′ terminal portion is complementary to the 3′ terminal portion of the DNA 5′ tail, under conditions permitting (i) the RNA strand to hybridize with the 5′ tail and (ii) the topoisomerase to covalently join the RNA strand and first DNA strand, thereby covalently joining the DNA strand to the RNA strand.
A method of obtaining a cDNA corresponding to a gene, the method comprising:
- (a) contacting a sequence-specific type I DNA topoisomerase in vitro with a double-stranded DNA whose first strand is to be covalently joined to an mRNA strand corresponding to the gene, wherein the first strand comprises a recognition sequence for the topoisomerase at a site whose complement on the second strand is located 3′ of the 5′ terminus of the second strand, so as to permit the topoisomerase to bind to the DNA and cleave the first strand thereof, thereby forming a topoisomerase/DNA complex wherein the DNA's second strand has a 5′ tail;
- (b) contacting the resulting complex in vitro with a 5′OH-containing mRNA strand corresponding to the gene and having a 5′ terminal portion complementary to the 3′ terminal portion of the DNA 5′ tail, under conditions permitting (i) the mRNA strand to hybridize with the 5′ tail and (ii) the topoisomerase to covalently join the mRNA strand and first DNA strand, thereby covalently joining the DNA strand to the mRNA strand;
- and
- (c) producing cDNA using as a template the covalently joined mRNA and DNA strands resulting from step (b), thereby obtaining a cDNA corresponding to the gene.
1 more element
Dependencies are inferred from the claim text.