Process for DNA replication
First Claim
1. A method for replication of a target region of a target DNA molecule comprising the steps of:
- (a) introducing a D-Loop into the target duplex DNA molecule at a first initiation point adjacent to the target region in a reaction mixture, wherein the step of introducing a D-loop is performed by hybridizing the duplex DNA molecule with a first oligonucleotide primer which is substantially complementary to the first initiation site;
(b) adding proteins to the reaction mixture to assemble a replisome at the D-loop; and
(c) providing DNA monomers and ATP to the replisome, whereby the target region is reproduced, and further comprising the step of introducing a second D-loop by hybridizing the duplex DNA molecule with a second oligonucleotide primer which is substantially complementary to a second initiation site, said target region lying between the first and second initiation sites.
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Accused Products
Abstract
A method is provided for replicating DNA, and in particular for replicating large segments of DNA. A primer is combined with a target DNA molecule to be replicated. The primer is designed to be at least partially homologous to a known site on the target DNA, and to create a D-loop when hybridized with that site. A replisome is then assembled at the D-loop, and this replisome creates a copy of the DNA, starting at the primer binding site. By utilizing two species of D-loop primers which bind to remote sites on the DNA flanking a region to be replicated, large sections of DNA can be replicated in a manner comparable to PCR. The replicated DNA can be analyzed to detect variations in the genetic sequence of the target, for linkage mapping and as a source of longer DNA molecules having a desired sequence.
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Citations
11 Claims
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1. A method for replication of a target region of a target DNA molecule comprising the steps of:
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(a) introducing a D-Loop into the target duplex DNA molecule at a first initiation point adjacent to the target region in a reaction mixture, wherein the step of introducing a D-loop is performed by hybridizing the duplex DNA molecule with a first oligonucleotide primer which is substantially complementary to the first initiation site;
(b) adding proteins to the reaction mixture to assemble a replisome at the D-loop; and
(c) providing DNA monomers and ATP to the replisome, whereby the target region is reproduced, and further comprising the step of introducing a second D-loop by hybridizing the duplex DNA molecule with a second oligonucleotide primer which is substantially complementary to a second initiation site, said target region lying between the first and second initiation sites. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification