Products and processes for multiplex nucleic acid identification

• US 10,604,791 B2
• Filed: 07/17/2012
• Issued: 03/31/2020
• Est. Priority Date: 05/19/2011
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Inventors: Honisch, Christiane, Van Den Boom, Dirk Johannes, Mosko, Michael, Nygren, Anders
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/551,486
• Publication Number:
  US 20130017960A1
• Time in Patent Office: 2,814 Days
• Field of Search: None
• 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 multiplex method for detecting the presence or absence of genetic variants of a plurality of target nucleic acid species in a composition comprising a sample, comprising:

(a) preparing a plurality of amplicons derived from a plurality of target nucleic acid species, or portions thereof, wherein nucleotide sequences of a target nucleic acid species comprise a first allele comprising a mutant sequence and a second allele comprising a wild-type sequence, wherein the first allele is of lower abundance and the second allele is of higher abundance and the lower abundance allele, if present, is about 1% or less of the target nucleic acid species;

(b) hybridizing the amplicons to a plurality of oligonucleotide species, wherein an oligonucleotide species comprising a hybridization sequence capable of specifically hybridizing under hybridization conditions to amplicons derived from the first allele and the second allele of a target nucleic acid species at a position 5′

to a single base position that differs between the first allele and the second allele of the target nucleic acid species, thereby generating hybridized oligonucleotides; and

(c) in a first container contacting the hybridized oligonucleotides with an extension composition comprising one, two or three terminating nucleotides specific for one or more of the first alleles and no terminating nucleotides specific for the second alleles under extension conditions;

wherein;

(i) the one, two or three terminating nucleotide or nucleotides each comprise biotin, and

(ii) oligonucleotides hybridized to a first allele of a target nucleic acid species, if present, are extended by a terminating nucleotide specific for one or more of the first alleles and oligonucleotides hybridized to a second allele of a target nucleic acid species are not extended by a terminating nucleotide specific for one or more of the first alleles, thereby generating extended oligonucleotides comprising a terminating nucleotide specific for the first alleles of the plurality of target nucleic acid species and not generating extended oligonucleotides for the second alleles;

(d) capturing the extended oligonucleotides to a solid phase comprising streptavidin or avidin;

(e) contacting the solid phase in (d) under conditions consisting of free biotin at a concentration of about 25 ug/ml to about 50 ug/ml and a temperature of about 90 degrees Celsius to about 95 degrees Celsius for about 1 to about 5 minutes, whereby a detectable amount of the extended oligonucleotides of the first allele for each nucleic acid species are released from the solid phase; and

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

thereby detecting the presence or absence of the first alleles of the plurality of target nucleic acid species.

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

of the region of the oligonucleotide species that hybridizes to an amplicon.

3. The method of claim 1, wherein the mutant sequence of a first allele comprises a somatic mutation.

4. The method of claim 1, wherein the terminating nucleotides consist of one terminating nucleotide.

5. The method of claim 1, wherein the terminating nucleotides consist of two terminating nucleotides.

6. The method of claim 1, wherein the terminating nucleotides consist of three terminating nucleotides.

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

8. The method of claim 1, wherein the plurality of target nucleic acid species is about 2 to about 20 target nucleic acid species.

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

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

11. The method of claim 1, comprising washing the solid phase after the extended oligonucleotides are captured.

12. The method of claim of 11, wherein the washing removes salts that produce interfering adducts in mass spectrometry analysis.

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

14. The method of claim 1, wherein a signal to noise ratio and/or a sensitivity for extending only a first allele is greater than a signal to noise ratio and/or a sensitivity for extending a first allele and a second allele.

15. The method of claim 1, wherein in (e) the temperature is about 90 degrees Celsius for about 5 minutes and the free biotin concentration is about 25 ug/ml.

16. The method of claim 1, wherein in (e) the temperature is about 95 degrees Celsius for about 5 minutes and the free biotin concentration is about 50 ug/ml.