Methods for attaching adapters to sample nucleic acids

Abstract

Methods of preparing double-stranded nucleic acids with single-stranded overhangs for amplification and sequencing are disclosed. Contacting a blunt-ended double-stranded nucleic acid molecules with Taq results in non-templated directed addition of a single nucleotide to the 3′ ends of the nucleic acid with A added most frequently followed by G followed by C and T. G tailing is sufficiently frequent that the efficiency of ligation of nucleic acid molecules to adapters can be significantly increased by including adapters tailed with T and C. The ligation efficiency can be increased even further with blunted-ended adapters to ligate to blunt-ended nucleic acid molecules that failed to undergo tailing.

1 . A method of preparing nucleic acids for analysis comprising;
 (a) blunt-ending double-stranded nucleic acids with single-stranded overhangs in a sample by the action of one or more enzymes providing a 5′-3′ polymerase activity and 3′-5′ proof reading activity, and four standard nucleotide types, wherein single-stranded overhangs with 5′ ends serve as templates for extension of a complementary strand by the polymerase activity and single-stranded overhangs with 3′ ends are digested by the proof reading activity producing blunt-ended nucleic acids;   (b) without separating the blunt-ended nucleic acids from other components of the sample, end-tailing the blunt-ended nucleic acids by action of a polymerase without a 3′-5′ proof reading function, which performs a non-template directed addition of a nucleotide to the 3′ ends of blunt-ended nucleic acids, wherein A is added preferentially to G preferentially to C or T;   (c) annealing the nucleic acids from step (b) with at least partially double-stranded adapters with a single nucleotide T or C overhangs at a 3′-end; and   (d) ligating the nucleic acids to the adapters.   
     
     
         2 . The method of  claim 1 , further comprising denaturing the one or more enzymes after step (a). 
     
     
         3 . The method of  claim 1 , further comprising contacting the sample with the one or more enzymes, the four standard nucleotide types and the polymerase without a 3′-5′ proof reading function. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein step (b) is performed at a higher temperature than step (a). 
     
     
         6 . The method of  claim 1 , wherein step (a) is performed at ambient temperature and step (b) at a temperature over 60° C. 
     
     
         7 . The method of  claim 1 , wherein the one or more enzymes are a polymerase with 5′-3′ polymerase activity and 3′-5′ proof reading activity. 
     
     
         8 . The method of  claim 1 , wherein the polymerase without a 3′-5′ proof reading function is a thermostable polymerase and the method further comprises increasing temperature of the sample after step (a) to inactivate the polymerase with 5′-3′ polymerase activity and 3′-5′ proof reading activity. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , further comprising contacting the sample with double-stranded blunt-ended adapters, which ligate with blunt-ended double-stranded nucleic acids which have not undergone the non-template directed addition of a nucleotide to the 3′ ends in the ligating step. 
     
     
         11 . The method of  claim 7 , wherein the polymerase with 5′-3′ polymerase activity and 3′-5′ proof reading activity is T4 polymerase or Klenow large fragment. 
     
     
         12 . The method of  claim 1 , wherein the polymerase without a 3′-5′ proof reading function is a Taq polymerase. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein for at least steps (a)-(d) no component is removed from the sample. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein at least partially double-stranded adapters with a single nucleotide T to a single nucleotide C has a molar ratio of 4:1 to 2:1. 
     
     
         17 . The method of  claim 16 , wherein the molar ratio of blunt-ended adapters to tailed adapters is 1:5 to 1:500. 
     
     
         18 . The method of  claim 1 , wherein at least 70% of the double-stranded nucleic acids in the sample are joined to adaptors. 
     
     
         19 - 35 . (canceled) 
     
     
         36 . A population of adapted nucleic acids produced by  claim 1 , the population comprising a plurality of nucleic acid molecules each of which comprises a nucleic acid fragment flanked on both sides by an adapter including a bar code with an A/T or G/C base pair between the nucleic acid fragment and adapter. 
     
     
         37 . The population of  claim 36 , wherein the plurality of nucleic acid molecules is at least 100,000 molecules. 
     
     
         38 . The population of  claim 36 , wherein the ratio of A/T base pairs to G/C base pairs is between 2:1 and 4:1. 
     
     
         39 . The population of  claim 36  wherein at least 99% of nucleic acid molecules in the population have a nucleic acid fragment flanked by adapters with different bar codes. 
     
     
         40 - 178 . (canceled)