Compositions and methods for detecting allelic variants
View Patent ↗The present invention provides compositions, methods, and kits for discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides compositions, methods, and kits for determining the presence and/or level (e.g., quantitating) of rare (e.g., mutant) allelic variants, such as single nucleotide polymorphisms (SNPs) or nucleotide insertions or deletions, in samples comprising abundant (e.g., wild-type) allelic variants with high sensitivity and/or specificity. As such, in certain embodiments, the present invention provides a highly selective method for the detection of somatic mutations, e.g., in samples containing abundant levels of a wild-type allele compared to very low levels of a mutant allele.
1. 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, said method comprising:
(a) forming a reaction mixture 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 wherein 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, wherein 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) a locus-specific primer that is complementary to a region of the target sequence, wherein the region of the target sequence complementary to the locus-specific primer is 3′ from the first allelic variant and on the opposite strand; and
(b) carrying out an amplification reaction on the reaction mixture using the locus-specific primer and the allele-specific primer to form an amplicon; and
(c) detecting the amplicon 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.
2. The method of claim 1 , wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the allele-specific nucleotide portion.
3. The method of claim 1 , wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the 5′-end and/or 3′-end of the allele-specific primer.
4. The method of claim 1 , wherein the allele-specific primer comprises two or more non-consecutive nucleic acid modifications.
5. The method of claim 1 , wherein the nucleic acid modification in the allele-specific primer is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
6. The method of claim 1 , wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at the allele-specific nucleotide portion.
7. The method of claim 1 , wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at an internal position in the allele-specific blocker probe.
8. The method of claim 1 , wherein the allele-specific blocker probe comprises two or more non-consecutive nucleic acid modifications.
9. The method of claim 1 , wherein the nucleic acid modification in the allele-specific blocker probe is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
10. The method of claim 1 , wherein the detector probe comprises a TaqMan® probe.
11. The method of claim 1 , wherein the nucleic acid sample is selected from the group consisting of blood, serum, plasma, fine needle aspirate, tumor tissue, and combinations thereof.
12. The method of claim 1 , wherein the first allelic variant is a mutant allele and the second allelic variant is the wild-type allele.
13. The method of claim 1 , wherein the method reduces the background signal of the second allelic variant during the amplification reaction.