Method to protect DNA ends

• US 20060024698A1
• Filed: 05/06/2005
• Published: 02/02/2006
• Est. Priority Date: 05/07/2004
• Status: Active Grant

• 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)
• Inventors: Tanaka, Masashi, Battista, John R., Jolivet, Edmond, Cox, Michael M., Saveliev, Sergei V., Harris, Dennis R., Eggington, Julie M.
• Granted Patent: US 7,211,393 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 the 3′

end of a DNA molecule from nuclease damage comprising the step of exposing the DNA molecule to an amount of a preparation of DdrA protein effective to decrease nuclease damage.

2. The method of claim 1, 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 2, wherein the method is used to join DNAs of at least 1 Kb each.

7. The method of claim 2, wherein the method reconstructs DNAs from mixtures of multiple DNA fragments of greater than 1 Kb.

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.

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.

17. A polynucleotide encoding the protein of claim 9.

16. A preparation of DdrA protein.