Fast hybridization for next generation sequencing target enrichment

Patent metadata

Field	Value
Publication number	US-9587268-B2
Application number	US-201414167513-A
Country	US
Kind code	B2
Filing date	Jan 29, 2014
Priority date	Jan 29, 2014
Publication date	Mar 7, 2017
Grant date	Mar 7, 2017

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.

First claim

What is claimed is:

1. A method for hybridizing a target nucleic acid to a bait nucleic acid, comprising
contacting a target nucleic acid with a bait nucleic acid in a composition and forming a hybridization mixture, said composition comprising
(i) a salt of a divalent cation that has a concentration in a range from about 100 mM to about 600 mM,
(ii) 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, and
(iii) a volume-excluding/thickening agent that has a concentration in a range from about 0.002% to about 0.1%, and

incubating the mixture for 8 hours or less,
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, methyl cellulose, and hydroxyl methyl cellulose.

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

3. The method of claim 1 , wherein the buffering agent has a concentration in a range from about 10 mM to about 40 mM and is selected from the group consisting of Tris, HEPES, TAPS, Tricene, Biceine, Bis-Tris, NaOH, KOH, TES, EPPS, and MOPS.

4. The method of claim 1 , wherein the divalent cation is magnesium.

5. The method of claim 4 , wherein the salt is MgCl 2 , the volume-excluding/thickening agent is HPMC, and the buffering agent is Tris.

6. The method of claim 1 , 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.

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

8. The method of claim 1 , wherein the incubating step is conducted at two different temperatures.

9. The method of claim 1 , wherein the incubating step is conducted at one temperature, and the time duration for the incubating step is less than 2 hours.

10. The method of claim 1 , wherein the time duration for the incubating step is less than 2 hours.

11. A method for hybridizing a target nucleic acid to a bait nucleic acid, comprising
contacting a target nucleic acid with a bait nucleic acid in a composition and forming a hybridization mixture, said composition comprising a salt of a divalent cation that has a concentration in a 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; and
incubating the mixture for 8 hours or less at two different temperatures, wherein the incubating step is cycled between said two different temperatures for 2 to 100 times.

12. The method of claim 11 , said composition further comprising a volume-excluding/thickening agent that has a concentration in the range from about 0.002% to about 15%.

13. The method of claim 12 , 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, methyl cellulose, and hydroxyl methyl cellulose.

14. The method of claim 13 , 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.

15. The method of claim 13 , wherein the salt is MgCl 2 , the volume-excluding/thickening agent is HPMC, and the buffering agent is Tris.

16. The method of claim 11 , wherein the divalent cation is selected from the group consisting of magnesium, calcium, manganese, cobalt, zinc, and cadmium.

17. The method of claim 11 , wherein the buffering agent has a concentration in a range from about 10 mM to about 40 mM and is selected from the group consisting of Tris, HEPES, TAPS, Tricene, Biceine, Bis-Tris, NaOH, KOH, TES, EPPS, and MOPS.

18. The method of claim 11 , wherein the divalent cation is magnesium.

19. The method of claim 11 , wherein the pH of the composition is about 7-11.

20. The method of claim 11 , wherein the time duration for the incubating step is less than 2 hours.

Assignees

• Agilent Technologies Inc
• Agilent Technologies Inc

Inventors

• Borns Michael

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2527/137

  Concentration of a component of medium · CPC title