CLOSED NUCLEIC ACID STRUCTURES
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 forming a closed nucleic acid structure from a target nucleic acid, comprising:
(a) contacting a target nucleic acid with a primer pair under PCR conditions, each of the primers having a 5′ phosphate group, thereby forming an amplified nucleic acid comprising a segment of the target nucleic acid flanked by the primers duplexed with their complementary segments;
(b) contacting the amplified nucleic acid with a terminal deoxynucleotidyl transferase (TdT) and a first deoxynucleotide thereby extending the 3′ ends of the amplified nucleic acid with a homo-oligomeric tail comprising the first deoxynucleotide;
(c) annealing an adaptor having a 5′ region and a 3′ region to the extended nucleic acid, the 5′ region of the adaptor having a 5′ phosphate group and comprising a stem-loop structure, and the 3′ region of the adaptor being complementary to the homo-oligomeric tail of the extended nucleic acid, wherein the 3′ region of the adaptor hybridizes to the homo-oligomeric tail thereby forming an adaptor-capped nucleic acid;
(d) contacting the adaptor-capped nucleic acid with the first deoxynucleotide and/or a second deoxynucleotide which is complementary to the first deoxynucleotide, and a DNA polymerase, thereby filling in any gap in the adaptor-capped nucleic acid with the first or the second deoxynucleotide; and
(e) contacting the closed nucleic acid structure with a ligase which seals nicks in the closed nucleic acid structure.
24 . The method of claim 23 , wherein the first deoxynucleotide is dATP, and the second deoxynucleotide is dTTP/dUTP.
25 . The method of claim 23 , wherein the target nucleic acid is a genomic DNA molecule.
26 . The method of claim 23 , wherein the target nucleic acid is a cDNA molecule.
27 . The method of claim 23 , wherein the target nucleic acid is a RNA molecule.
28 . The method of claim 23 , wherein the ligase is a T4 DNA ligase.
29 . The method of claim 23 , wherein each of the primers has a 5′ segment of at least 5 nucleobase units.
30 . The method of claim 23 , wherein each of the primers has a 3′ segment of at least 10 nucleobase units.
31 . The method of claim 23 , wherein the target nucleic acid is a PCR product.
32 . The method of claim further comprising sequencing the closed nucleic acid thereby determining the sequence of the target nucleic acid.