| 摘要 |
<p>Detecting a target nucleic acid, comprises a primer-mediated amplification of at least one target nucleic acid, where at least one of the primers used for amplification is a sequence-tag-primer, which exhibits a non-hybridizing part at its 5'-end; (b) contacting the partially double-stranded amplified product from step (a) with nucleotides, a nicking endonuclease and a polymerase; (c) isothermal amplification of the sequence-tags produced in step (a) by one or more repetition of a cycle; and (d) specific detection of the sequence tags amplified in step (c). Detecting a target nucleic acid, comprises a primer-mediated amplification of at least a target nucleic acid, where at least one of the primers used for the amplification is a sequence-tag-primer, which exhibits a non-hybridizing part at its 5'-end, the non-hybridizing part exhibits a first sequence that produces an interface at the newly synthesized strand, which is complementary to the first sequence, for a nicking endonuclease during the amplification, and further 5' of the first sequence comprises a second sequence that produces a sequence tag at a newly synthesized strand, which is complementary to the second sequence, during the amplification; (b) contacting the partially double-stranded amplified product of step (a) with nucleotides, a nicking endonuclease and a polymerase, where the polymerase has a strand-displacement-activity and no 5'-3' exonuclease activity; (c) isothermal amplification of the sequence-tags produced in step (a) by one or more repetition of a cycle that comprises the steps of (i) making a nick in the interface inserted in the step (a) by the endonuclease of step (b), and filling the nicks generated in the step (i) at free 3'-end with complementary nucleotides using the polymerase of step (b) with simultaneous displacement of the sequence tags from the double strand; and (d) specific detection of the sequence tags amplified in step (c).</p> |
