Granted Patent B2

US 12,630,865 · App. 18/139,303 · Granted May 19, 2026

Inventors: Christiane Honisch (La Jolla, CA); Dirk Van Den Boom (Encinitas, CA); Michael Mosko (Poway, CA); Anders Nygren (San Diego, CA)

Assignee: AGENA BIOSCIENCE, INC.

C12Q1/6823C12Q1/6809C12Q1/6853C12Q1/6858

Abstract

Claims (31)

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 and wherein the first variant is a lower abundance variant and the second variant is a higher abundance variant;

(b) hybridizing the amplicons to oligonucleotide species, wherein each oligonucleotide species has a mass that detectably differs from the masses of the other oligonucleotide species and each oligonucleotide species specifically hybridizes to an amplicon derived from a target nucleic acid species, thereby generating hybridized oligonucleotide species;

(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 oligonucleotide 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 the terminating nucleotides consist of one terminating nucleotide, two terminating nucleotides or three terminating nucleotides.

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

4 . The method of claim 1 , wherein the plurality of target nucleic acid species is about 2 to about 500 target nucleic acid species.

5 . The method of claim 4 , wherein the plurality of target nucleic acid species is about 5, 10, 25, 50, 75, 100, 150 or 200 target nucleic acid species.

6 . The method of claim 1 , wherein the capture agent comprises biotin or a biotin analogue.

7 . The method of claim 6 , wherein the solid phase comprises avidin or a modified form of avidin or streptavidin or a modified form of streptavidin.

8 . The method of claim 7 , wherein releasing the extended oligonucleotide species is by a releasing agent comprising free biotin or a biotin analogue.

9 . 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 and wherein the first variant is a lower abundance variant and the second variant is a higher abundance variant;

(b) hybridizing the amplicons to oligonucleotide species, wherein each oligonucleotide species comprises a mass distinguishable tag located 5′ of the hybridization sequence, the mass of the mass distinguishable tag of each oligonucleotide species detectably differs from the masses of mass distinguishable tags of the other oligonucleotide species, and each mass distinguishable tag specifically corresponds to an amplicon derived from a target nucleic acid species, thereby generating hybridized oligonucleotide species;

(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 oligonucleotide 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 distinguishable tags associated with or cleaved from the extended oligonucleotide species captured to the solid phase in (d), or detecting the mass distinguishable tags associated with or cleaved from the 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.

10 . The method of claim 9 , wherein the mass distinguishable tag is cleaved from the extended oligonucleotide species.

11 . The method of claim 9 , wherein the mass distinguishable tag is a nucleotide compomer.

12 . The method of claim 11 , wherein the nucleotide compomer is about 1 to 30, 5 to 35, 10 to 30, 15 to 30, 20 to 35, 25 to 35, 30 to 40, 35 to 45, 40 to 50, or 25 to 50 nucleotides in length.

13 . The method of claim 9 , wherein the terminating nucleotides independently are selected from ddATP, ddGTP, ddCTP, ddTTP and ddUTP.

14 . The method of claim 9 , wherein the plurality of target nucleic acid species is about 2 to about 500 target nucleic acid species.

15 . The method of claim 14 , wherein the plurality of target nucleic acid species is about 5, 10, 25, 50, 75, 100, 150 or 200 target nucleic acid species.

Assignments (4)

Continuity (5)

References Cited (392)