Method and Kits for Repairing Nucleic Acid Sequences

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.

1 . A DNA repair kit, comprising:
 a thermally labile DNA polymerase having 5′-3′ exonuclease activity; and   a thermally labile DNA ligase.   
     
     
         2 . The method of  claim 1 , wherein the thermally labile DNA polymerase and the thermally labile DNA ligase are substantially inactivated by incubation at a temperature between about 42° C. and about 75° C. 
     
     
         3 . The DNA repair kit of  claim 1 , wherein the DNA polymerase is  Escherichia coli  DNA polymerase I. 
     
     
         4 . The DNA repair kit of  claim 1 , further comprising at least one additional DNA repair enzyme. 
     
     
         5 . The DNA repair kit of  claim 1 , further comprising at least one of 8-oxoguanine DNA glycosylase, an endonuclease IV, an endonuclease VIII, or a T4 endonuclease V. 
     
     
         6 . The DNA repair kit of  claim 1 , wherein the DNA polymerase and the DNA ligase are a premixed single solution. 
     
     
         7 . The DNA repair kit of  claim 1 , further comprising a solution containing a glycosylase enzyme and/or an endonuclease enzyme. 
     
     
         8 . A DNA repair kit, comprising a combination of enzymes, wherein the combination exhibits N-glycosylase, AP-lyase, 5′-3′ DNA polymerase, and 5′-3′ exonuclease nick translation activities, and wherein the combination of enzymes is heat-inactivatable. 
     
     
         9 . A DNA repair kit, comprising:
 a DNA glycosylase;   a DNA endonuclease;   a DNA polymerase I; and   a T4 DNA ligase.   
     
     
         10 . The DNA repair kit of  claim 9 , wherein the DNA glycosylase comprises 8-oxoguanine DNA glycosylase. 
     
     
         11 . The DNA repair kit of  claim 9 , wherein the endonuclease comprises edonuclease IV, endonuclease VIII, T4 endonuclease V, or a combination thereof. 
     
     
         12 . The DNA repair kit of  claim 9 , further comprising a buffer solution having a pH of about 7.9. 
     
     
         13 . The DNA repair kit of  claim 9 , wherein the DNA polymerase I comprises both 5′-3′ DNA polymerase activity and 5′-3′ exonuclease activity. 
     
     
         14 . A method for repairing damaged DNA, comprising:
 incubating damaged DNA with a DNA repair enzyme blend at a first temperature to generate repaired DNA, wherein the DNA repair enzyme blend comprises a thermally labile DNA polymerase having 5′-3′ exonuclease activity and a thermally labile DNA ligase.   
     
     
         15 . The method of  claim 14 , further comprising inactivating the enzyme blend by incubating the repaired DNA and the DNA repair enzyme blend at a second temperature, wherein the second temperature is higher than the first temperature. 
     
     
         16 . The method of  claim 14 , further comprising amplifying the repaired DNA. 
     
     
         17 . The method of  claim 14 , further comprising performing a polymerase chain reaction on the repaired DNA. 
     
     
         18 . The method of  claim 14 , further comprising analyzing the repaired DNA for comparison to a database of known DNA samples. 
     
     
         19 . The method of  claim 14 , wherein the second temperature is in the range of about 42° C. to about 75° C. 
     
     
         20 . A method for DNA repair, comprising:
 removing a base from a damaged site on a DNA strand via a glycosylase;   nicking the DNA strand at the damaged site via an endonuclease;   translating the nick down the DNA strand via a DNA polymerase having an associated 5′-3′ exonuclease activity; and   sealing the nick with a ligase.   
     
     
         21 . The method of  claim 20 , wherein the DNA polymerase and the ligase are thermally labile. 
     
     
         22 . The method of  claim 20 , wherein the DNA polymerase comprises both a 5′-3′ DNA polymerase activity and a 5′-3′ DNA exonuclease activity.