Repair of Nucleic Acids for Improved Amplification

Abstract

Methods and compositions are provided for repairing a polynucleotide so that it can be synthesized efficiently with improved fidelity and yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI. The reaction mixture may further contain an AP endonuclease and a polymerase. These enzymes are optionally selected according to their ability to withstand high temperatures so they can be included in an amplification mixture. The reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture.

1 . A method for enhancing at least one of fidelity and yield of an amplification product of a damaged polynucleotide, comprising the steps of:
 (a) incubating the polynucleotide containing one or more damaged sites with a reaction mixture comprising an effective amount of a ligase in the absence of ATP, and in the absence of Endonuclease (Endo) VI, for repairing the damaged sites in the polynucleotide; and   (b) amplifying the polynucleotide in the reaction mixture.   
     
     
         2 . A method according to  claim 1 , wherein the reaction mixture in step (a) further comprises a polymerase and an AP endonuclease. 
     
     
         3 . A method according to  claim 2 , wherein the AP endonuclease is a class II AP endonuclease. 
     
     
         4 . A method according to  claim 3 , wherein the AP endonuclease is selected from the group consisting of a T4 endonuclease, an  E. coli  endonuclease, Tth Endo IV, and human AP endonuclease. 
     
     
         5 . A method according to  claim 3 , wherein the AP endonuclease is  E. coli  Endo IV. 
     
     
         6 . A method according to  claim 2 , wherein the polymerase is selected from the group consisting of Taq DNA polymerase, Bst DNA polymerase, T4 DNA polymerase, T7 DNA polymerase,  E. coli  DNA polymerase I and an archaeal DNA polymerase or modifications thereof. 
     
     
         7 . A method according to  claim 2 , wherein the polymerase is an archaeal DNA polymerase and the archaeal polymerase is selected from Pfu, Vent®, Deep Vent®, 9° North, and GBD DNA polymerase. 
     
     
         8 . A method according to  claim 2 , wherein the polynucleotide is DNA and the reaction mixture in step (a) comprises 1-100 units of the AP endonuclease, 0.05-0.25 units of the polymerase and 5-500 units of the ligase. 
     
     
         9 . A method according to  claim 1 , wherein the reaction mixture in (a) further comprises T4 pyrimidine dimer glycosylase (pdg). 
     
     
         10 . A method according to  claim 1 , wherein the reaction mixture in step (a) further comprises [fapy]-DNA glycosylase (Fpg). 
     
     
         11 . A method according to  claim 1 , wherein the reaction mixture in (a) further comprises at least one of UvrA, UvrB, UvrC, UvrD and Cho. 
     
     
         12 . A method according to  claim 1 , wherein the reaction mixture in (a) further comprises at least one glycosylase/lyase 
     
     
         13 . A method according to  claim 12 , wherein the at least one glycosylase/lyase is selected from the group consisting of Endo III, Endo VIII, Fpg, OGGI, and T4 pdg. 
     
     
         14 . A method according to  claim 1 , wherein the reaction mixture in (a) further comprises at least one glycosylase. 
     
     
         15 . A method according to  claim 1 , wherein the at least one glycosylase is selected from the group consisting of UDG, AlkA and Aag. 
     
     
         14 . A method according to  claim 2 , wherein the polymerase is a Bst DNA polymerase, the AP endonuclease is Endo IV, the ligase is Taq ligase, the reaction mixture in (a) further comprising one or more glycosylases and one or more glycosylase/lyases. 
     
     
         15 . A method according to  claim 1 , wherein the amplification is PCR amplification, helicase-dependent amplification, transcription-mediated amplification, strand-displacement amplification, rolling circle amplification or whole genome amplification. 
     
     
         16 . A method according to  claim 14 , wherein the at least one glycosylase is UDG. 
     
     
         17 . A method according to  claim 2 , wherein the polymerase is an  E. coli  Y family DNA polymerase.