Preparation of adapter-ligated amplicons

Abstract

The present invention is directed to novel methods and kits to be employed for preparing adapter-ligated amplicons or a sequencing library of a target DNA, respectively.

1 . A kit for preparing adapter-ligated PCR amplicons, comprising
 (i) at least one polynucleotide kinase,   (ii) at least one DNA ligase,   (iii) DNA adapter molecules,   (vi) a reaction buffer configured to simultaneously allow the enzymatic functioning of said at least one polynucleotide kinase and said at least one DNA ligase, wherein the reaction buffer may contain,
 i) phosphate donors, 
 ii) ions, 
 iii) NAD as co-factor 
   (v) a manual.   
     
     
         2 . The kit of  claim 1 , wherein said DNA polynucleotide kinase is selected from the group consisting of: T4 polynucleotide kinase, Klenow fragment, TopTaq polymerase, Tfl-Polymerase, Tma-Polymerase, Tne-Polymerase and Tth-Polymerase. 
     
     
         3 . The kit of  claim 1 , wherein said DNA ligase is selected from the group consisting of:
 T4 DNA ligase, T3 DNA ligase, T7 DNA ligase,  E. coli  DNA Ligase, Taq DNA ligase, and 9° N DNA ligase.   
     
     
         4 . The kit of  claim 2 , wherein said DNA ligase is selected from the group consisting of:
 T4 DNA ligase, T3 DNA ligase, T7 DNA ligase,  E. coli  DNA Ligase, Taq DNA ligase, and 9° N DNA ligase.   
     
     
         5 . The kit of  claim 4 , wherein said DNA polynucleotide kinase is T4 polynucleotide kinase and said DNA ligase is T4 DNA ligase. 
     
     
         6 . The kit of  claim 1 , wherein the DNA adapter molecules comprise a T-overhang.