Method and kits for repairing nucleic acid sequences

Patent metadata

Field	Value
Publication number	US-9574183-B2
Application number	US-201213344504-A
Country	US
Kind code	B2
Filing date	Jan 5, 2012
Priority date	Jul 8, 2008
Publication date	Feb 21, 2017
Grant date	Feb 21, 2017

Abstract

Methods and kits for DNA repair are provided. The methods and kits described herein repair multiple types of DNA damage. The kit may include a plurality of enzymes to repair a greater variety of lesions than any single enzyme is capable of repairing. Repair of damaged DNA may include releasing damaged bases from the DNA strand, nicking the DNA at the damaged sites, translating the nicks via 5′-3′ exonuclease activity, and sealing the nicks. The enzymes employed in the repair process may then be heat-inactivated, thereby obviating a purification process. The repaired DNA may then be analyzed using a variety of DNA analysis methods.

First claim

The invention claimed is:

1. A method for repairing damaged genomic DNA, comprising:
incubating damaged genomic DNA with a premixed DNA repair enzyme blend solution at a first temperature to generate repaired genomic DNA, wherein the DNA repair enzyme blend comprises a DNA polymerase having 5′-3′ exonuclease activity and a DNA ligase, and wherein a ratio of units of the DNA ligase to units of the DNA polymerase is approximately 40:1 in the premixed DNA repair enzyme blend solution; and
inactivating the DNA repair enzyme blend by incubating the repaired genomic DNA and the DNA repair enzyme blend at a second temperature without denaturing the repaired genomic DNA, wherein the second temperature is higher than the first temperature.

2. The method of claim 1 , further comprising amplifying the repaired genomic DNA.

3. The method of claim 1 , further comprising performing a polymerase chain reaction on the repaired genomic DNA.

4. The method of claim 1 , further comprising analyzing the repaired genomic DNA for comparison to a database of known DNA samples.

5. The method of claim 1 , wherein the second temperature is in the range of about 42° C. to about 75° C.

6. A method for genomic DNA repair, comprising:
in a single reaction utilizing a DNA repair enzyme blend comprising a glycosylase, an endonuclease, a DNA polymerase, and a ligase at a first temperature to generate repaired genomic DNA by simultaneously performing the steps of:
removing a base from a damaged site on a genomic DNA strand via the glycosylase;
nicking the genomic DNA strand at the damaged site via the endonuclease;
translating the nick down the genomic DNA strand via the DNA polymerase having an associated 5′-3′ exonuclease activity; and
sealing the nick with the ligase; and
inactivating the DNA repair enzyme blend by incubating the repaired genomic DNA and the DNA repair enzyme blend at a second temperature without denaturing the repaired genomic DNA, wherein the second temperature is higher than the first temperature.

7. The method of claim 6 , wherein the DNA polymerase and the ligase are thermally labile.

8. The method of claim 6 , wherein the DNA polymerase comprises both a 5′-3′ DNA polymerase activity and a 5′-3′ DNA exonuclease activity.

9. The method of claim 1 , wherein the damaged genomic DNA comprises oxidatively or ultravioletly damaged DNA.

10. The method of claim 2 , wherein the repaired genomic DNA is not purified prior to amplification.

11. The method of claim 3 , wherein the repaired genomic DNA is not purified prior to the polymerase chain reaction.

12. The method of claim 4 , wherein the repaired genomic DNA is not purified prior to analysis.

13. A method for repairing damaged genomic DNA, comprising:
incubating damaged genomic DNA with a premixed DNA repair enzyme blend solution at a first temperature to generate repaired genomic DNA, wherein the DNA repair enzyme blend simultaneously repairs different types of DNA damage, and the DNA repair enzyme blend comprises at least a DNA polymerase having 5′-3′ exonuclease activity, DNA ligase, a glycosylase, and an endonuclease; and
inactivating the DNA repair enzyme blend by incubating the repaired genomic DNA and the DNA repair enzyme blend at a second temperature without denaturing the repaired genomic DNA, wherein the second temperature is higher than the first temperature.

14. The method of claim 13 , comprising amplifying the repaired genomic DNA without purifying the repaired genomic DNA.

15. The method of claim 13 , comprising performing a polymerase chain reaction on the repaired genomic DNA without purifying the repaired genomic DNA.

16. The method of claim 13 , comprising analyzing the repaired genomic DNA for comparison to a database of known DNA samples without purifying the repaired genomic DNA.

Assignees

• Nelson John Richard
• Gao Wei
• Gen Electric

Inventors

• Nelson John Richard
• Gao Wei

Classifications

• C12N9/93

  Ligases (6) · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title