Nucleic Acid Sample Preparation

Abstract

This invention relates to the preparation of nucleic acid samples for analysis. The invention may be particularly useful for single stranded samples. Embodiments of the invention involve the attachment of double stranded or hairpin oligonucleotides using template independent polymerase enzymes in the preparation of nucleic acid sequencing libraries.

1 .- 24 . (canceled) 
     
     
         25 . A method comprising: contacting a single stranded oligonucleotide with an enzyme, wherein said enzyme catalyzes an addition of a nucleotide to an end of said single stranded oligonucleotide in a template independent manner, wherein said nucleotide comprises a 5′-triphosphate. 
     
     
         26 . The method of  claim 25 , wherein said enzyme comprises a terminal transferase (TdT). 
     
     
         27 . The method of  claim 26 , wherein said terminal transferase comprises a terminal deoxynucleotidyl transferase (TdT), a polyadenylate polymerase (PAP) or a poly(U)polymerase (PUP). 
     
     
         28 . The method of  claim 25 , wherein said nucleotide comprises a plurality of nucleotides. 
     
     
         29 . The method of  claim 25 , wherein said single stranded oligonucleotide is produced by at least one of (i) a bisulfite treatment; (ii) a chemical or enzymatic cleavage; or (iii) use of an enzyme that is a restriction endonuclease to form said single stranded oligonucleotide. 
     
     
         30 . The method of  claim 25 , wherein a second oligonucleotide strand comprises said nucleotide. 
     
     
         31 . The method of  claim 30 , further comprising associating at least a portion of said single stranded oligonucleotide with at least a portion of said second oligonucleotide strand to form a double-stranded oligonucleotide. 
     
     
         32 . The method of  claim 31 , wherein said second oligonucleotide strand comprises a hairpin. 
     
     
         33 . The method of  claim 32 , wherein said hairpin comprises an extendable 3′-end. 
     
     
         34 . The method of  claim 31 , wherein said second oligonucleotide strand is associated with a solid support. 
     
     
         35 . The method of  claim 34 , wherein said second oligonucleotide strand comprises a moiety for attachment to said solid support. 
     
     
         36 . The method of  claim 31 , wherein said associating comprises ligation between said single stranded oligonucleotide and said second oligonucleotide strand. 
     
     
         37 . The method of  claim 25 , wherein said single stranded oligonucleotide comprises a plurality of single stranded nucleotides. 
     
     
         38 . The method of  claim 37 , wherein said plurality of single stranded nucleotides comprises a whole genome library or a loci specific library. 
     
     
         39 . The method of  claim 37 , wherein said plurality of single stranded nucleotides are different. 
     
     
         40 . The method of  claim 25 , wherein said single stranded oligonucleotide comprises DNA. 
     
     
         41 . The method of  claim 25 , wherein said single stranded oligonucleotide comprises a modified base. 
     
     
         42 . The method of  claim 25 , wherein said enzyme is a non-naturally occurring enzyme.