Method to protect DNA ends

• US 7,211,393 B2
• Filed: 05/06/2005
• Issued: 05/01/2007
• Est. Priority Date: 05/07/2004
• Status: Expired due to Fees

• Current Assignee: Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System), Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System), Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Tanaka, Masashi, Battista, John R., Jolivet, Edmond, Cox, Michael M., Saveliev, Sergei V., Harris, Dennis R., Eggington, Julie M.
• Primary Examiner(s): Fredman; Jeffrey
• Assistant Examiner(s): Babic; Christopher M.
• Application Number:
  US11/123,701
• Publication Number:
  US 20060024698A1
• Time in Patent Office: 725 Days
• Field of Search: None
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

A method of protecting the 3′ end of a DNA molecule from nuclease damage is disclosed. In one embodiment, the method comprises the step of exposing the DNA molecule to a preparation of DdrA protein.

Claims

1. A method of protecting a 3′

end of a DNA molecule from exonuclease damage comprising the step of exposing the DNA molecule to an amount of a pure preparation of DdrA protein effective to decrease exonuclease damage in relation to natural exonuclease damage that would occur in the absence of DdrA, wherein the DdrA protein consists of SEQ ID NO;

2.

8. The method of claim 1 additionally including the step of exposing the DNA molecule to DrSSB.

9. The method of claim 1 wherein the DNA is between 0.1–

100 nanomolar.

10. The method of claim 1 wherein the DdrA protein is between 1 nanomolar–

100 micromolar.

11. The method of claim 10 wherein the concentration of DdrA is 1–

10 micromolar.

12. The method of claim 1 wherein the DNA is protected for at least 30 minutes.

2. A method of protecting a 3′

end of a DNA molecule from exonuclease damage comprising the step of exposing the DNA molecule to an amount of a PURE preparation of DdrA protein effective to decrease exonuclease damage in relation to natural exonuclease damage that would occur in the absence of DdrA, wherein the DdrA protein consisting of SEQ ID NO;

2, and wherein the DdrA protein is incubated with at least a first and a second linear duplex DNA each with a 3′

-ending strand and a 5′

-ending strand, wherein the first linear duplex DNAs comprises an end that is complementary to an end of the second linear duplex DNA.

3. The method of claim 2 further comprising the step of exposing the first and second DNA molecule to a exonuclease, wherein the DdrA protein protects the 3′

ending strands while allowing the 5′

ending strands to be degraded, thereby producing single-stranded extensions with 3′

ends on both first and second DNAs.

4. The method of claim 3 further comprising the steps of annealing the single stranded extensions of the first and second DNAs, thereby producing a joined first and second DNA.

5. The method of claim 4 further comprising the steps of processing the joined DNA with a nuclease, a DNA polymerase, and a DNA ligase to make the joined DNA contiguous.

6. The method of claim 5, wherein the method is used to join the first and second DNAs of at least 1 Kb each.

7. The method of claim 6, wherein the method reconstructs the first and second DNAs from mixtures of multiple DNA fragments of greater than 1 Kb.

13. The method of claim 2 additionally including the step of exposing the DNA molecule to DrSSB.

14. The method of claim 5 wherein DNA is between 0.1–

100 nanomolar.

15. The method of claim 5 wherein the DdrA protein is between 1 nanomolar–

100 micromolar.