Isothermal nucleic acid amplification

摘要

The disclosure relates to an isothermal process for amplifying a double-stranded nucleic acid target molecule. The process comprises providing an upstream primer, a downstream primer, a strand invasion system and an oligonucleotide. The upstream and downstream primers are not substrates for the strand invasion system during the amplification process and do not amplify the target molecule independently of the strand invasion system, and the oligonucleotide is a substrate for the strand invasion system.

主权项

1. An isothermal process for amplifying a double-stranded nucleic acid target molecule comprising the following steps:
(a) providing:
(i) a recombinase;(ii) an upstream nucleic acid primer and a downstream nucleic acid primer that do not bind the double-stranded nucleic acid target molecule, are not substrates for the recombinase, and do not amplify the target molecule independently of the recombinase, wherein each of said upstream and downstream nucleic acid primers comprises a single-stranded DNA molecule of less than 25 nucleotides, wherein at least a portion of each nucleic acid primer is complementary to a sequence of the target molecule; and(iii) an oligonucleotide comprising a single-stranded DNA molecule of at least 30 nucleotides, at least 24 of said nucleotides of said oligonucleotide are complementary to the target molecule in a region between upstream and downstream nucleic acid primer binding sites, and at least a portion of the oligonucleotide is substrate for the recombinase; (b) contacting the oligonucleotide with the double-stranded target molecule in the presence of the recombinase and allowing said oligonucleotide to invade the target molecule so that the region of the target molecule complementary to the oligonucleotide and regions adjacent to a terminus of the complementary region are rendered single-stranded to form a first strand and a second strand; (c) applying the upstream nucleic acid primer to the first strand of the target molecule at a region adjacent to and within 20 nucleotides of the region complementary to the oligonucleotide and extending the 3′ end of the upstream nucleic acid primer with polymerase and dNTPs to produce a double-stranded nucleic acid target molecule; (d) applying the downstream primer to the second strand of the target molecule at a region adjacent to and within 20 nucleotides of the region complementary to the oligonucleotide and extending the 3′ end of the downstream nucleic acid primer with polymerase and dNTPs to produce a further double-stranded nucleic acid target molecule; and (e) continuing the reaction through repetition of (b) to (d).