Methods for Adding Adapters to Nucleic Acids and Compositions for Practicing the Same

Abstract

Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template ribonucleic acid (RNA), a template switch oligonucleotide including a 3′ hybridization domain and a sequencing platform adapter construct, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template RNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.

1 - 22 . (canceled) 
     
     
         23 . A method comprising:
 adding a nucleic acid sequence to a 3′-end of a precursor nucleic acid to produce a template nucleic acid;   combining:
 the template nucleic acid; 
 a primer comprising a domain that hybridizes to the template nucleic acid; 
 a template switch oligonucleotide; 
 a polymerase; and 
 dNTPs, 
   into a reaction mixture under conditions sufficient to produce a complex comprising the template nucleic acid and the template switch oligonucleotide each hybridized to a single product nucleic acid polymerized from the dNTPs in a template switching reaction.   
     
     
         24 . The method according to  claim 23 , wherein the precursor nucleic acid is:
 a small target nucleic acid of 100 nucleotides or less in length; or   a large target nucleic acid of 100 nucleotides or greater in length,   present in a nucleic acid mixture.   
     
     
         25 . The method according to  claim 23 , wherein the precursor nucleic acid is a polyadenylated nucleic acid or a non-polyadenylated nucleic acid present in a nucleic acid mixture. 
     
     
         26 . The method according to  claim 23 , further comprising:
 fragmenting a nucleic acid to produce the precursor nucleic acid.   
     
     
         27 . The method according to  claim 23 , wherein adding the nucleic acid sequence comprises ligating the nucleic acid sequence to the 3′ end the precursor nucleic acid. 
     
     
         28 . The method according to  claim 23 , further comprising amplifying single product nucleic acid. 
     
     
         29 . The method according to  claim 28 , wherein the amplifying comprises contacting the single product nucleic acid with a first amplification primer comprising at least a portion of a sequence present in the primer and a second amplification primer comprising at least a portion of a sequence present in the template switch oligonucleotide. 
     
     
         30 . The method according to  claim 23 , wherein at least one of the primer, the template switch oligonucleotide, the first amplification primer, the second amplification primer or a combination thereof includes a barcode.