Multiplex methods for detection and quantification of minor variants

• US 10,640,817 B2
• Filed: 04/22/2016
• Issued: 05/05/2020
• Est. Priority Date: 04/24/2015
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Inventors: Nygren, Anders Olof Herman
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/568,701
• Publication Number:
  US 20180298433A1
• Time in Patent Office: 1,474 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Provided herein are multiplex methods for detecting the presence or absence and amount of variants of a plurality of target nucleic acid species having low-abundance variants and high-abundance variants.

Claims

1. A multiplex method for detecting the presence or absence of variants of a plurality of nucleic acid species, comprising:

(a) preparing a plurality of amplicons derived from a plurality of target nucleic acid species, or portions thereof, wherein each target nucleic acid species comprises a low-abundance variant and a high-abundance variant;

(b) in a single reaction hybridizing the amplicons to a plurality of oligonucleotide species, each oligonucleotide species specifically corresponds to amplicons of a target nucleic acid species, wherein (i) an oligonucleotide species hybridizes to amplicons derived from the low-abundance variant and the high-abundance variant of a target nucleic acid species at a location whereby a single base position that differs between the low-abundance variant and the high-abundance variant of the target nucleic acid species is 3′

to the hybridized oligonucleotide species, (ii) the nucleotide at the single base position is the same or different for each of the high-abundance variants of the plurality of target nucleic acid species, (iii) the nucleotide at the single base position is the same for each of the low-abundance variants of the plurality of target nucleic acid species and (iv) none of the nucleotides at the single base positions for the high-abundance variants of the plurality of target nucleic acid species are the same as the nucleotide at the single base position for the low-abundance variants of the plurality of target nucleic acid species;

thereby generating hybridized oligonucleotides; and

(c) contacting the hybridized oligonucleotides with an extension composition comprising a terminating nucleotide specific for the low-abundance variants and one, two or three terminating nucleotides specific for one or more of the high-abundance variants under extension conditions;

wherein;

(i) the terminating nucleotides comprises a capture agent, (ii) the concentration of the one, two or three terminating nucleotides specific for one or more high-abundance variants are each at a concentration of from 0.1% to 30% relative to the concentration of the terminating nucleotide specific for the low-abundance variants, (iii) the extension conditions comprise multiple thermal cycles, thereby generating extended oligonucleotides comprising a terminating nucleotide specific for the low-abundance variants of the plurality of target nucleic acid species and extended oligonucleotides comprising a terminating nucleotide specific for the high-abundance variants of the plurality of target nucleic acid species;

(d) contacting the extended oligonucleotides with a solid phase under conditions in which the capture agent interacts with the solid phase, thereby capturing the extended oligonucleotides onto the solid phase, wherein the solid phase comprises a binding partner of the capture agent in (c);

(e) releasing the extended oligonucleotides in (d) by contacting the solid phase at elevated temperature conditions with a competitor, wherein the competitor comprises the free form of the capture agent that interacts with the solid phase in (d); and

(f) detecting the extended oligonucleotides released in (e);

thereby detecting the presence or absence of the variants of a plurality of nucleic acid species.

2. The method of claim 1, wherein the variants are single nucleotide polymorphism (SNP) variants, the low-abundance variant is a lower-abundance allele and the high-abundance variant is a higher-abundance allele.

3. The method of claim 1, wherein the low-abundance variant is a mutant allele and the high-abundance variant is the wild type of the allele.

4. The method of claim 1, wherein the low-abundance variant has one or more insertions or a deletions and the high-abundance variant does not have one or more insertions or deletions.

5. The method of claim 1, wherein the low-abundance variant is present in a copy number that is 2% or less than the copy number of the high-abundance variant.

6. The method of claim 1, wherein the one or more terminating nucleotides specific for the high-abundance variants consist of one terminating nucleotide, two different terminating nucleotides or three different terminating nucleotides.

7. The method of claim 1, wherein the terminating nucleotides independently are selected from ddATP, ddGTP, ddCTP, ddTTP and ddUTP or comprise one or more acyclic terminators.

8. The method of claim 1, wherein the terminating nucleotides comprise one or more acyclic terminators.

9. The method of claim 1, wherein the concentration of each of the one, two or three terminating nucleotides specific for the high-abundance variants is at a concentration of from 0.5% to 10% relative to the concentration of the specific terminating nucleotide for the low-abundance variants.

10. The method of claim 1, wherein the concentration of each of the one, two or three terminating nucleotides specific for the high-abundance variants is at a concentration of from 1% to 2% relative to the concentration of the specific terminating nucleotide for the low-abundance variants.

11. The method of claim 1, wherein the amplicons produced in (a) are contacted with an agent that removes terminal phosphates from any nucleotides not incorporated into the amplicons.

12. The method of claim 1, wherein the plurality of target nucleic acid species is 10 or more target nucleic acid species or 20 or more target nucleic acid species.

13. The method of claim 1, wherein the capture agent comprises biotin, the solid phase comprises streptavidin and the competitor comprises free biotin.

14. The method of claim 13, wherein the competitor is at a concentration from about 10 ug/ml to about 100 ug/ml and the elevated temperature conditions are about 80°

C. to about 100°

C.

15. The method of claim 1, wherein in the absence of detection of an extended oligonucleotide comprising a terminating nucleotide specific for a low-abundance variant, the detection of an extended oligonucleotide comprising a terminating nucleotide specific for a high-abundance variant provides a positive control for a false negative result.

16. The method of claim 1, wherein each extended oligonucleotide comprises a fluorescent label and a terminating nucleotide comprises the fluorescent label.

17. The method of claim 1, wherein each extended oligonucleotide comprises a mass label and the mass label is a mass distinguishable tag that is located 5′

of the region of the oligonucleotide species that hybridizes to an amplicon or the terminating nucleotide comprises the mass-distinguishable tag.

18. The method of claim 17, wherein detection is by mass spectrometry.

19. The method of claim 1, further comprising determining the amount of a low-abundance variant relative to the amount of a high-abundance variant for each target nucleic acid species which comprises normalization based on the ratio of the concentration of the terminating nucleotide specific for the low-abundance variant and the concentration of the terminating nucleotide specific for the high-abundance variant.

20. A multiplex method for detecting the presence or absence and amount of variants of a plurality of nucleic acid species, comprising:

(a) preparing a plurality of amplicons derived from a plurality of target nucleic acid species, or portions thereof, wherein each target nucleic acid species comprises a low-abundance variant and a high-abundance variant;

(b) in a single reaction hybridizing the amplicons to a plurality of oligonucleotide species, each oligonucleotide species specifically corresponds to amplicons of a target nucleic acid species, wherein (i) an oligonucleotide species hybridizes to amplicons derived from the low-abundance variant and the high-abundance variant of a target nucleic acid species at a location whereby a single base position that differs between the low-abundance variant and the high-abundance variant of the target nucleic acid species is 3′

to the hybridized oligonucleotide species, (ii) the nucleotide at the single base position is the same or different for each of the high-abundance variants of the plurality of target nucleic acid species, (iii) the nucleotide at the single base position is the same for each of the low-abundance variants of the plurality of target nucleic acid species and (iv) none of the nucleotides at the single base positions for the high-abundance variants of the plurality of target nucleic acid species are the same as the nucleotide at the single base position for the low-abundance variants of the plurality of target nucleic acid species;

thereby generating hybridized oligonucleotides; and

(c) contacting the hybridized oligonucleotides with an extension composition comprising a terminating nucleotide specific for the low-abundance variants and one, two or three terminating nucleotides specific for one or more of the high-abundance variants under extension conditions;

wherein;

(i) the terminating nucleotides comprises a capture agent, (ii) to the concentration of the one, two or three terminating nucleotides specific for one or more high-abundance variants are each at a concentration of from 0.1% to 30% relative to the concentration of the terminating nucleotide specific for the low-abundance variants, (iii) the extension conditions comprise multiple thermal cycles, thereby generating extended oligonucleotides comprising a terminating nucleotide specific for the low-abundance variants of the plurality of target nucleic acid species and extended oligonucleotides comprising a terminating nucleotide specific for the high-abundance variants of the plurality of target nucleic acid species;

(d) contacting the extended oligonucleotides with a solid phase under conditions in which the capture agent interacts with the solid phase, thereby capturing the extended oligonucleotides onto the solid phase, wherein the solid phase comprises a binding partner of the capture agent in (c);

(e) releasing the extended oligonucleotides in (d) by contacting the solid phase at elevated temperature conditions with a competitor, wherein the competitor comprises the free form of the capture agent that interacts with the solid phase in (d);

(f) detecting the extended oligonucleotides released in (e);

thereby detecting the presence or absence of the variants of the plurality of nucleic acid species, and

(g) for variants of the plurality of the nucleic acid species detected as present in (f), determining the amount of the low-abundance variant relative to the amount of the high-abundance variant for each target nucleic acid species.