Method for molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase
AssigneeSloan-Kettering Institute for Cancer Research
InventorStewart Shuman
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Abstract
This invention provides a modified vaccinia topoisomerase enzyme containing an affinity tag which is capable of facilitating purification of protein-DNA complexes away from unbound DNA. This invention further provides a modified sequence specific topoisomerase enzyme. This invention provides a method of ligating duplex DNAs, a method of molecular cloning of DNA, a method of synthesizing polynucleotides, and a method of gene targeting. Lastly, this invention provides a recombinant DNA molecule composed of segments of DNA which have been joined ex vivo by the use of a sequence specific topoisomerase and which has the capacity to transform a suitable host cell comprising a DNA sequence encoding polypeptide activity.
Claims9
A method of ligating a donor duplex DNA to acceptor DNA near each end of the donor duplex DNA, which method comprises the steps of:
- (a) incubating a sequence-specific topoisomerase with a donor duplex DNA, each of whose strands has near its 3′ end a cleavage site specifically recognized by the topoisomerase, whereby the topoisomerase binds to the donor duplex DNA near each end thereof and then specifically cleaves the donor duplex DNA at each cleavage site, so as to form a complex comprising the donor duplex DNA having near each end thereof a bound topoisomerase and a 5′ single-stranded tail;
- and
- (b) incubating the complex formed in step (a) with a 5′ hydroxyl-terminated acceptor DNA having a single-stranded tail complementary to the 5′ single-stranded tail of the complex, so as to ligate the donor duplex DNA to the acceptor DNA near each end of the donor duplex DNA.
The method of claim 7, wherein the acceptor DNA comprises an affinity label.
Dependencies are inferred from the claim text.