Methods, compositions, and kits for detecting allelic variants

• US 10,081,833 B2
• Filed: 09/16/2015
• Issued: 09/25/2018
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Caifu, Tan, Ruoying
• Primary Examiner(s): Thomas, David C
• Application Number:
  US14/856,448
• Publication Number:
  US 20160076090A1
• Time in Patent Office: 1,105 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

Claims

1. A method for detecting a first allelic variant of a target sequence in a nucleic acid sample suspected of comprising at least a second allelic variant of the target sequence, comprising:

a) forming a first reaction mixture by combining;

i) the nucleic acid sample;

ii) a first allele-specific primer, wherein an allele-specific nucleotide portion of the first allele-specific primer is complementary to the first allelic variant of the target sequence, wherein the first allele-specific primer does not have a locked nucleic acid (LNA) base;

iii) a first allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the first allele-specific primer, and wherein the first allele-specific blocker probe comprises a minor groove binder at the 3′

-end, the 5′

-end and/or at an internal position within said allele-specific blocker probe;

iv) a first 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; and

v) a first detector probe;

b) carrying out an amplification reaction on the first reaction mixture using the first locus-specific primer and the first allele-specific primer to form a first amplicon; and

c) detecting the first amplicon by detecting a change in a detectable property of the first detector probe, thereby detecting the first allelic variant of the target gene in the nucleic acid sample.

2. The method of claim 1, further comprising using the change in a detectable property of the first detector probe to quantitate the first allelic variant.

3. The method of claim 1, further comprising:

d) forming a second reaction mixture by combining;

i) the nucleic acid sample;

ii) a second allele-specific primer, wherein an allele-specific nucleotide portion of the second allele-specific primer is complementary to the second allelic variant of the target sequence;

iii) a second allele-specific blocker probe that is complementary to a region of the target sequence comprising the first allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the second allele-specific primer, and wherein the second allele-specific blocker probe comprises a minor groove binder at the 3′

-end, the 5′

-end and/or at an internal position within said allele-specific blocker probe;

iv) a second locus-specific primer that is complementary to a region of the target sequence that is 3′

from the second allelic variant and on the opposite strand; and

v) a second detector probe;

e) carrying out an amplification reaction on the second reaction mixture using the second allele-specific primer and the locus-specific primer, to form a second amplicon; and

f) detecting the second amplicon by detecting a change in a detectable property of the detector probe, thereby detecting the second allelic variant of the target gene in the nucleic acid sample.

4. The method of claim 3, further comprising comparing the change in a detectable property of the first detector probe in the first reaction mixture to the change in a detectable property of the second detector probe in the second reaction mixture.

5. The method of claim 3, wherein said first, second or first and second allele-specific primer and/or said first, second, or first and second allele-specific blocker probe comprises at least one modified base.

6. The method of claim 5, wherein said modified base is an 8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A), cytosine (C), guanine (G), or thymine (T).

7. The method of claim 5, wherein said modified base is a fdU or iso dC base.

8. The method of claim 5, wherein said modified base is any modified base that increases the Tm between matched and mismatched target sequences or nucleotides.

9. The method of claim 5, wherein said modified base is located at (a) the 3′

-end, (b) the 5′

-end, (c) at an internal position or at any combination of (a), (b) or (c) within said allele-specific primer and/or allele-specific blocker probe.

10. The method of claim 5, wherein the specificity of said detecting is improved at least 2 fold by the inclusion of said modified base in said first, second or first and second allele-specific primer and/or said first, second, or first and second allele-specific blocker probe as compared to when it is not.

11. The method of claim 3, wherein said carrying out an amplification reaction comprises a 2-stage cycling protocol.

12. The method of claim 11, wherein the number of cycles in the first stage of said 2-stage cycling protocol comprises fewer cycles than the number of cycles used in the second stage.

13. The method of claim 11, wherein said number of cycles in the first stage is about 90% fewer cycles than said number of cycles in the second stage.

14. The method of claim 11, wherein said number of cycles in the first stage is between 3-7 cycles and said number of cycles in the second stage is between 42-48 cycles.

15. The method of claim 11, wherein the annealing/extension temperature used during the first cycling stage of said 2-stage cycling protocol is between 1-3°

C. lower than the annealing/extension temperature used during the second stage.

16. The method of claim 11, wherein said annealing/extension temperature used during the first cycling stage of said 2-stage cycling protocol is between 56-59°

C. and said annealing/extension temperature used during said second stage is between 60-62°

C.

17. The method of claim 1, wherein said step (a) is preceded by a pre-amplification step.

18. The method of claim 17, wherein said pre-amplification step comprises a multiplex amplification reaction that uses at least two complete sets of allele-specific primers and locus-specific primers, wherein each set is suitable or operative for amplifying a specific polynucleotide of interest.

19. The method of claim 18, wherein the products of said multiplex amplification reaction are divided into secondary single-plex amplification reactions, wherein each single-plex amplification reaction contains at least one primer set previously used in said multiplex reaction.

20. The method of claim 18, wherein said multiplex amplification reaction further comprises a plurality of allele-specific blocker probes.