FAST HYBRIDIZATION FOR NEXT GENERATION SEQUENCING TARGET ENRICHMENT

• US 20150211047A1
• Filed: 01/29/2014
• Published: 07/30/2015
• Est. Priority Date: 01/29/2014
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Borns, Michael
• Granted Patent: US 9,587,268 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

The present invention relates to compositions and methods of target enrichment or selection of nucleic acids using hybridization, which can be used in, e.g., next-generation sequencing.

Claims

1. A composition for nucleic acid hybridization, comprising a salt of a divalent cation that has a concentration in the range from about 100 mM to about 600 mM and a buffering agent, wherein said salt and said buffering agent are present in a molar ratio of about 2.5:

1 to about 60;

1.

2. The composition of claim 1, further comprising a volume-excluding/thickening agent that has a concentration in the range from about 0.002% to about 15% w/w.

3. The composition of claim 1, wherein the buffering agent has a concentration in the range from about 10 mM to about 40 mM.

4. The composition of claim 1, wherein the divalent cation is selected from the group consisting of magnesium, calcium, manganese, cobalt, zinc, and cadmium.

5. The composition of claim 1, wherein the volume-excluding/thickening agent is selected from the group consisting of hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methycellulose, and hydroxyl methyl cellulose.

6. The composition of claim 5, wherein the volume-excluding/thickening agent has a concentration in the range from about 0.002% to about 0.1% w/w.

7. The composition of claim 1, wherein the volume-excluding/thickening agent is selected from the group consisting of Dextran and polyethylene glycol (PEG).

8. The composition of claim 1, wherein the buffering agent is selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, NaOH, KOH, TES, EPPS, and MOPS.

9. The composition of claim 1, wherein the divalent cation is magnesium.

10. The composition of claim 9, wherein the salt is MgCl₂, the volume-excluding/thickening agent is HPMC, and the buffering agent is Tris.

11. The composition of claim 10, wherein said salt has a concentration of about 308 mM, said HPMC has a concentration of about 0.00834%, and said buffering agent has a concentration of about 20 mM.

12. The composition of claim 1, wherein the pH of the composition is about 7-11.

13. A buffer concentrate for preparing a hybridization solution, wherein dilution by a factor ranging from 2-fold to 20-fold yields the composition of claim 1.

14. The buffer concentrate of claim 13, wherein dilution by a factor of 5-fold yields said composition.

15. A kit for nucleic acid hybridization, comprising the buffer concentrate of claim 13 and packaging material therefor.

16. A method for nucleic acid hybridization of a target nucleic acid and a bait nucleic acid, comprising

contacting a target nucleic acid and a bait nucleic acid with a composition of claim 1 to form a hybridization mixture;

denaturing the hybridization mixture at a first temperature for a first period of time;

blocking the mixture at a second temperature for a second period of time;

incubating the mixture (i) at a third temperature for a third period of time and then (ii) at a fourth temperature for a fourth period of time, and

repeating the incubating step for one or more times.

17. The method of claim 16, further comprising, after the repeating step, holding the mixture at a fifth temperature.

18. The method of claim 16, wherein the incubating step is carried out 20-100 times.

19. The method of claim 16, wherein the third temperature and the fourth temperature are different, and the time duration for the incubating step and repeating step is 3 hours or less.

20. The method of claim 16, wherein third temperature and the fourth temperature are the same, and the time duration for the incubating step and repeating step is about 1-4 hours.