PRODUCTS AND PROCESSES FOR MULTIPLEX NUCLEIC ACID IDENTIFICATION

• US 20130017960A1
• Filed: 07/17/2012
• Published: 01/17/2013
• Est. Priority Date: 05/19/2011
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Honisch, Christiane, Van Den Boom, Dirk J., Mosko, Michael, Nygren, Anders
• Granted Patent: US 10,604,791 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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 2563/167 Mass label
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Provided herein are products and processes for detecting the presence or absence of multiple target nucleic acids. Certain methods include amplifying the target nucleic acids, or portion thereof; extending oligonucleotides that specifically hybridize to the amplicons, where the extended oligonucleotides include a capture agent; capturing the extended oligonucleotides to a solid phase via the capture agent; releasing the extended oligonucleotide by competition with a competitor; detecting the extended oligonucleotide, and thereby determining the presence or absence of each target nucleic acid by the presence or absence of the extended oligonucleotide.

Claims

1. A method for detecting the presence, absence or amount of a plurality of genetic variants in a composition, 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 first variant and a second variant;

(b) hybridizing the amplicons to oligonucleotide species, wherein each oligonucleotide species hybridizes to an amplicon derived from a target nucleic acid species, thereby generating hybridized oligonucleotide species; and

(c) contacting the hybridized oligonucleotide species with an extension composition comprising one or more terminating nucleotides under extension conditions;

wherein;

(i) at least one of the one or more terminating nucleotide comprises a capture agent, and

(ii) the hybridized oligonucleotide species that hybridize to the first variant are extended by a terminating nucleotide and the hybridized oligonucleotide species that hybridize to the second variant are not extended by a terminating nucleotide, thereby generating extended oliognucleotide species;

(d) capturing the extended oligonucleotide species to a solid phase that captures the capture agent;

(e) releasing the extended oligonucleotide species bound to the solid phase in (d) from the solid phase; and

(f) detecting the mass of each extended oligonucleotide species released from the solid phase in (e) by mass spectrometry;

whereby the presence, absence or amount of the genetic variants is detected.

2. The method of claim 1, wherein each oligonucleotide species comprises a mass distinguishable tag located 5′

of the hybridization sequence.

3. The method of claim 1, wherein the first variant is a lower abundance variation and the second variant is a higher abundance variation.

4. The method of claim 1, wherein the genetic variants are single nucleotide polymorphism (SNP) variants, the first variant is a lower abundance allele and the second variant is a higher abundance allele.

5. The method of claim 1, wherein the one or more terminating nucleotides consist of one terminating nucleotide.

6. The method of claim 1, wherein the one or more terminating nucleotides consist of two terminating nucleotides.

7. The method of claim 1, wherein the one or more terminating nucleotides consist of three terminating nucleotides.

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

9. The method of claim 1, wherein the extension composition comprises a non-terminating nucleotide.

10. The method of claim 1, wherein the extension composition comprises one or more extension nucleotides, which extension nucleotides comprise no capture agent.

11. The method of claim 1, wherein releasing the extended oligonucleotide species comprises contacting the solid phase with a releasing agent.

12. The method of claim 11 wherein the capture agent comprises biotin or a biotin analogue, the solid phase comprises streptavidin and the releasing agent comprises free biotin or a biotin analogue.

13. The method of claim 11 wherein the releasing agent has a higher affinity for the solid phase than the capture agent.

14. The method of claim 1 wherein releasing the extended oligonucleotide species in (e) comprises heating from about 60°

C. to about 100°

C.

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

16. The method of claim 1, wherein the plurality of target nucleic acid species is 200 or more target nucleic acid species.

17. The method of claim 1, wherein the plurality of target nucleic acid species is 200 to 300 target nucleic acid species.

18. The method of claim 1, wherein the extension conditions in (c) comprise cycling 20 to 300 times.

19. The method of claim 1, wherein the extension conditions in (c) comprise cycling 200 to 300 times.

20. The method of claim 1, comprising washing the solid phase after the extended oligonucleotide species is captured.

21. The method of claim of 20, wherein the washing removes salts that produce interfering adducts in mass spectrometry analysis.

22. The method of claim of 1, wherein extended oligonucleotides are not contacted with an ion exchange resin.

23. The method of claim 1, wherein a signal to noise ratio and/or a sensitivity for extending only a mutant allele is greater than a signal to noise ratio and/or a sensitivity for extending a wild type and a mutant allele.

24. The method of claim 1, wherein the extended oligonucleotide species of the second variant is not detected.

25. The method of claim 11, wherein the releasing agent is added at a concentration from about 10 to about 100 ug/ml.

26. The method of claim 1, wherein the composition comprises a synthetic template and the amount and/or percentage of a first variant in the composition is determined wherein the synthetic template comprises a variant different than in the first variant and second variant and hybridizes to the same oligonucleotides species.

27. A method for determining the presence or absence of a plurality of target nucleic acids in a composition, which comprises:

(a) preparing amplicons of the target nucleic acids by amplifying the target nucleic acids, or portions thereof, under amplification conditions;

(b) contacting the amplicons in solution with a set of oligonucleotides under hybridization conditions, wherein;

(i) each oligonucleotide in the set comprises a hybridization sequence capable of specifically hybridizing to one amplicon under the hybridization conditions when the amplicon is present in the solution,

(ii) each oligonucleotide in the set comprises a mass distinguishable tag located 5′

of the hybridization sequence,

(iii) the mass of the mass distinguishable tag of one oligonucleotide detectably differs from the masses of mass distinguishable tags of the other oligonucleotides in the set; and

(iv) each mass distinguishable tag specifically corresponds to a specific amplicon and thereby specifically corresponds to a specific target nucleic acid;

(c) generating extended oligonucleotides that comprise a capture agent by extending oligonucleotides hybridized to the amplicons by one or more nucleotides, wherein one of the one of more nucleotides is a terminating nucleotide and one or more of the nucleotides added to the oligonucleotides comprises the capture agent;

(d) contacting the extended oligonucleotides with a solid phase under conditions in which the capture agent interacts with the solid phase;

(e) releasing the mass distinguishable tags in association with the extended oligonucleotides that have interacted with the solid phase from the solid phase by competition with a competitor; and

(f) detecting the mass distinguishable tags released in (e) by mass spectrometry;

whereby the presence or absence of each target nucleic acid is determined by the presence or absence of the corresponding mass distinguishable tag.

28. The method of claim 27, wherein competition with a competitor comprises contacting the solid phase with a competitor.

29. The method of claim 27, wherein the competitor consists of free capture agent, or a competing fragment or multimer thereof.

30. The method of 27, wherein the detecting in (f) comprises a signal to noise ratio greater than the signal to noise ratio for a method in which releasing does not comprise competition with a competitor.