| 主权项 |
1. A method of forming a closed nucleic acid structure comprising a target nucleic acid, the method comprising:
(a) providing a denatured target nucleic acid, each strand of the denatured target nucleic acid having a 5′ phosphate and a 3′ segment; (b) contacting the strands of the denatured target nucleic acid with a pair of 3′ blocked oligonucleotides, each oligonucleotide having a 3′ segment and a 5′ segment, the 3′ segments being complementary to the 3′ segments of the strands of the denatured target nucleic acid; (c) annealing the 3′ segments of the 3′ blocked oligonucleotides to the 3′ segments of the denatured target nucleic acid strands, whereby each 3′ blocked oligonucleotide provides a template for extension of the denatured target nucleic acid strand to which it is annealed; (d) extending the denatured target nucleic acid strands to form 3′ extended nucleic acid strands, each 3′ extended nucleic acid strand having from 5′ to 3′ a 5′ phosphate group, a strand of the target nucleic acid, a 3′ extension complementary to the 5′ segment of a 3′ blocked oligonucleotide, and a 3′ hydroxyl group; (e) contacting a duplex nucleic acid intermediate formed by annealing of the 3′ extended nucleic acid strands to one another with a pair of stem-loop adaptors, each adaptor having a 3′ segment and a 5′ segment, the 5′ segments each having a 5′ phosphate group and a sequence capable of forming a stem-loop structure and the 3′ segments being complementary to the 3′ extensions of the duplex nucleic acid intermediate and ending in a 3′ hydroxyl group; (f) annealing the 3′ segments of the stem-loop adaptors to the 3′ extensions of the duplex target nucleic acid; and (g) providing a ligase that seals nicks between the ends of the stem-loop adaptors and the ends of duplex target nucleic acid, thereby forming a closed nucleic acid structure including the two strands of the target nucleic acid joined to one another by stem-loop structures. |
