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 - 45 . (canceled) 
     
     
         46 . A method comprising:
 (a) providing a precursor ribonucleic acid (RNA);   (b) adding a plurality of non-templated nucleotides to an end of the precursor RNA to produce a template RNA;   (c) combining:
 the template RNA; 
 a template switch oligonucleotide (TSO) comprising a first nucleotide sequence domain; 
 a polymerase having terminal transferase activity; 
 a first strand primer comprising a first domain that hybridizes to the non-templated nucleotides in the template RNA and a second domain having a nucleotide sequence that is different from the first nucleotide sequence domain present in the TSO; and 
 dNTPs; 
   in a reaction mixture under conditions sufficient to produce a product nucleic acid comprising the template RNA and the TSO hybridized to adjacent regions of a single product nucleic acid comprising the first strand primer and a region polymerized from the dNTPs by the polymerase; and   (d) amplifying the product nucleic acid with a forward primer and a reverse primer,   wherein each of the forward primer and the reverse primer comprises a sequencing platform adapter construct comprising at least a portion of a capture sequence that is utilized by a sequencing platform,   wherein the capture sequence specifically hybridizes to a surface-attached sequencing platform oligonucleotide on the sequencing platform, and   wherein the forward and reverse primers are different and hybridize to different sequences of the product nucleic acid.   
     
     
         47 . The method according to  claim 46 , further comprising sequencing the amplified product nucleic acid by the sequencing platform that comprises the surface-attached sequencing platform oligonucleotide that captures the at least portion of the capture sequence of the sequencing platform adapter construct. 
     
     
         48 . The method according to  claim 46 , wherein the non-templated nucleotides are added to the 3′ end of the precursor RNA. 
     
     
         49 . The method according to  claim 46 , wherein the non-templated nucleotides are added in an enzymatic reaction. 
     
     
         50 . The method according to  claim 46 , wherein the non-templated nucleotides comprise a polyadenylation (poly A) sequence. 
     
     
         51 . The method according to  claim 46 , wherein the precursor RNA is a non-polyadenylated RNA. 
     
     
         52 . The method according to  claim 51 , wherein the non-polyadenylated RNA is selected from the group consisting of a microRNA, a siRNA, and a small RNA. 
     
     
         53 . The method according to  claim 46 , wherein the polymerase is a reverse transcriptase. 
     
     
         54 . The method according to  claim 46 , wherein the template switch oligonucleotide, the first strand primer, or both the template switch oligonucleotide and the first strand primer comprise a sequencing platform adapter construct that is utilized by a sequencing platform. 
     
     
         55 . The method according to  claim 54 , wherein the sequencing platform adapter construct comprises a nucleic acid domain selected from the group consisting of: a domain that specifically hybridizes to the surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof. 
     
     
         56 . A kit, the kit comprising:
 a) a template switch oligonucleotide (TSO) comprising a 3′ hybridization domain;   b) a first polymerase;   c) a first strand primer comprising a first domain that hybridizes to a template RNA;   d) dNTPs; and   e) a second polymerase.   
     
     
         57 . The kit according to  claim 56 , wherein the first polymerase further comprises terminal transferase activity and template switching activity 
     
     
         58 . The kit according to  claim 56 , wherein the first polymerase is a reverse transcriptase. 
     
     
         59 . The kit according to  claim 58 , wherein the reverse transcriptase is a MMLV reverse transcriptase. 
     
     
         60 . The kit according to  claim 56 , wherein the second polymerase adds dNTPs to the 3′ end of a precursor RNA to produce the template RNA.