| 摘要 |
A method for detecting or quantitating a first allelic variant of a target sequence in a nucleic acid sample suspected of having at least a second allelic variant of the target sequence is disclosed. The a reaction mixture is formed by combining (i) the nucleic acid sample; (ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the allele-specific primer is complementary to the first allelic variant of the target sequence, and the allele-specific primer comprises at least one nucleic acid modification; (iii) an allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant and the allele-specific blocker probe comprises a non-extendable 3’-hexanediol, blocker moiety and at least one nucleic acid modification; (iv) a detector probe; and (v) locus-specific primer that is complementary to a region of the target sequence where the region of the target sequence complementary to the locus-specific primer is 3’ from the first allelic variant; and on the opposite strand. Then an amplification reaction on the reaction mixture using the locus-specific primer and the allele-specific primer to form an amplicon is carried out. The amplicon is detected by detecting a change in a detectable property of the detector probe, thereby detecting the first allelic variant of the target gene in the nucleic acid sample. A composition comprises (a) an allele-specific primer, wherein an allele-specific nucleotide portion of the allele-specific primer is complementary to a first allelic variant of a target sequence and the allele-specific primer comprises at least one nucleic acid modification; and (b) an allele-specific blocker probe that is complementary to a region of the target sequence comprising a second allelic variant, and the allele-specific blocker probe comprises a non-extendable 3’-hexanediol, blocker moiety and at least one nucleic acid modification. The composition can further comprise (c) a detector probe; and/or (d) a locus-specific primer that is complementary to a region of the target sequence that is 3’ from the first allelic variant and on the opposite strand. A reaction mixture comprising the above described composition and (e) a nucleic acid molecule is also disclosed. |
