| 主权项 |
1. A method for enriching and optionally detecting a target nucleic acid with at least one variant nucleotide from a nucleic acid population in a sample, said method comprising:
(a) treating the sample with an enriching primer and an amplification primer for a first strand of the target nucleic acid sequence to create a mixture of duplexes
wherein the mixture of duplexes comprises the enriching primer and the amplification primer annealed to the target nucleic acid under hybridising conditions,wherein the nucleotide sequence of said enriching primer is such that it is substantially complementary to a diagnostic region where the variant nucleotide is locatedbut wherein a nucleotide at, or within 1, 2, or 3 nucleotides of the 3′ terminus is complementary to the corresponding normal nucleotide,and wherein the amplification primer is annealed to the target nucleic acid such that the 3′ end of the amplification primer is upstream of the 5′ end of the enriching primer; (b) maintaining the mixture of step (a) under extension conditions, which comprise nucleoside triphosphates and a nucleic acid polymerase which does not have strand displacement activity, to extend the annealed primers, if extendable, to synthesize primer extension products; (c) repeating steps (a) and (b) in an amplification reaction which is PCR; and (d) optionally detecting the enriched target nucleic acid, wherein extension of the enriching primer occurs when it is annealed to the diagnostic region of the target nucleic acid having the normal nucleotide to synthesize an enriching primer extension product, wherein the enriching primer extension product is not fully digested or displaced by the nucleic acid polymerase, whereby the enriching primer extension product thereby blocks the extension initiated from the amplification primer when the amplification primer is annealed to the target nucleic acid having the normal nucleotide, wherein the annealed enriching primer is not extendable when it anneals to the diagnostic region containing the variant nucleotide, whereby the enriching primer is dissociated from the target sequence under the extension conditions, thereby allowing extension of the amplification primer to pass through the diagnostic region containing the variant nucleotide leading to exponential amplification of the target nucleic acid containing the variant nucleotide, and wherein the enriching primer comprises a moiety that renders the extension product of the enriching primer unsuitable for an exponential amplification, thereby permitting preferential exponential amplification of the target nucleic acid having the variant nucleotide. |
