Process for DNA replication

• US 6,699,693 B1
• Filed: 10/26/2001
• Issued: 03/02/2004
• Est. Priority Date: 02/04/1999
• Status: Expired due to Fees

• Current Assignee: Sloan-Kettering Institute For Cancer Research (Memorial Sloan-Kettering Cancer Center)
• Original Assignee: Sloan-Kettering Institute For Cancer Research (Memorial Sloan-Kettering Cancer Center)
• Inventors: Marians, Kenneth, Liu, Joing
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/890,829
• Time in Patent Office: 858 Days
• Field of Search: 435/6, 435/7.32, 435/91.1, 435/91.2, 435/183, 436/94, 536/23.1, 536/23.7, 536/23.72, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/149 Replicase based amplification, e.g. Q beta replicase

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.

Claims

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.

2. The method of claim 1, wherein the first oligonucleotide primer has a length of from 20 to 50 bases.

3. The method of claim 1, wherein the first oligonucleotide primer comprises a detectable label or capture moiety.

4. The method of claim 1, wherein the first and second oligonucleotide primers each have a length of from 20 to 50 bases.

5. The method of claim 1, wherein at least one of the oligonucleotide primers comprises a detectable label or capture moiety.

6. The method of claim 1, wherein the replication is performed in a supporting matrix.

7. The method of claim 1, wherein the replisome is assembled via the action of primosomal proteins, single-stranded DNA-binding protein and the DNA polymerase III holoenzyme.

8. The method of claim 7, wherein the primosomal proteins includes a mutant PriA protein which lacks ATPase and helicase functionality.

9. The method of claim 3, wherein the replication is performed in a supporting matrix.

10. The method of claim 3, wherein the replisome is assembled via the action of primosomal proteins, single-strand binding protein and holoenzyme III.

11. The method of claim 10, wherein the primosomal proteins includes a mutant PriA protein which lacks ATPase and helicase functionality.