Closed nucleic acid structures

Abstract

The invention provides compositions and methods for making closed nucleic acid structures in which one or both strands are continuous. The closed nucleic acid structures can be used as sequencing templates among other applications.

1 - 22 . (canceled) 
     
     
         23 . A method of synthesizing a double-stranded nucleic acid comprising the steps of:
 (a) contacting a target nucleic acid with a terminal deoxynucleotidyl transferase and a first deoxynucleotide thereby extending a 3′ end of the target nucleic acid with a homo-oligomeric tail comprising the first deoxynucleotide;   (b) contacting the target nucleic acid with a primer having a 3′ target-binding segment complementary to the homo-oligomeric tail, whereby the target nucleic acid forms a template for extension from the primer;   (c) forming a first extended nucleic acid strand duplexed to the target nucleic acid, the first extended strand comprising from 5′-3′ the primer and a segment complementary to the target nucleic acid and non-templated nucleotides;   (d) contacting the first extended nucleic acid strand with an adapter complementary to the non-templated nucleotides; and   (e) further extending the extended strand with the adapter forming a template for the further extension thereby forming a double-stranded nucleic acid comprising the extended stranded duplexed to the target nucleic acid.   
     
     
         24 . The method  23 , further comprising
 (f) forming a second extended nucleic acid strand duplexed to the first extended strand, the second extended strand comprising from 5′-3′ the adapter, a segment complementary to the first extended strand and having a common sequence with the target nucleic acid, and a segment complementary to the primer, the 5′ end of the second extended strand being the phosphorylated 5′ end of the second primer and the 3′ end of the second extended strand having a 3′ hydroxyl group;   (g) denaturing the second extended strand from the first extended strand and contacting the second extended strand with a bridging oligonucleotide having a 5′ segment and a 3′ segment, the 5′ segment being complementary to a segment at the 5′ end of the second extended strand and the 3′ segment being complementary to a segment at the 3′ end of the second extended strand;   (h) annealing the 5′ segment of the bridging oligonucleotide to the 5′ segment of the second extended strand and the 3′ segment of the bridging oligonucleotide to the 3′ segment of the second extended strand, thereby forming a partially duplex, two-stranded intermediate, wherein the 5′ phosphate group of the second extended strand is separated by a nick from the 3′ hydroxyl group of the second extended strand; and   (i) providing a ligase that seals the nick between the 5′ phosphate and 3′ hydroxyl groups of the second extended strand, thereby forming a closed nucleic acid structure.   
     
     
         25 . The method of  claim 23 , further comprising amplifying the double-stranded target nucleic acid. 
     
     
         26 . The method of  claim 23 , further comprising sequencing the double-stranded target nucleic acid. 
     
     
         27 . The method of  claim 23 , wherein the target nucleic acid is a genomic DNA molecule. 
     
     
         28 . The method of  claim 23 , wherein the target nucleic acid is a cDNA molecule. 
     
     
         29 . The method of  claim 23 , wherein the target nucleic acid is an RNA molecule. 
     
     
         30 . The method of  claim 23 , wherein the target nucleic acid is a PCR product. 
     
     
         31 . The method of  claim 23 , wherein the target nucleic acid is single-stranded. 
     
     
         32 . The method of  claim 23 , wherein the target nucleic acid is part of a duplex. 
     
     
         33 . The method of  claim 32 , wherein the extension of the primer displaces the complementary strand of the duplex.