HIGH EFFICIENCY MULTIPLEXED NUCLEIC ACID CAPTURE IN A STRUCTURED MICROENVIRONMENT

• US 20150140553A1
• Filed: 11/05/2014
• Published: 05/21/2015
• Est. Priority Date: 11/15/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Ji, Hanlee P., Cushing, Anna McGinty, Lau, Billy Tsz Cheong
• Granted Patent: US 9,689,027 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: B01L 3/5021 Test tubes specially adapted for centrifugation purposes centrifuges B04B5/04
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2523/32 Centrifugation
• CPC Class Codes: C12Q 2527/156 Permeability

Abstract

Provided herein is a method for sample analysis. In some embodiments, the method may involve: a) enzymatically attaching a reactive group to nucleic acid molecules in a sample; b) covalently reacting the reactive group with surface exposed reactive sites on a porous support, thereby covalently tethering the nucleic acid molecules to the porous support; c) performing a primer extension reaction using the tethered nucleic acid molecules as a template to produce primer extension products; and d) eluting the primer extension products from the porous support, while leaving the tethered nucleic acid molecules tethered to the porous support.

Claims

1. A method for sample analysis, comprising:

a) enzymatically attaching a reactive group to nucleic acid molecules in a sample;

b) covalently reacting said reactive group with reactive sites in a porous support, thereby covalently tethering said nucleic acid molecules to said porous support;

c) performing a primer extension reaction using said tethered nucleic acid molecules as a template to produce primer extension products; and

d) eluting the primer extension products from the porous support, while leaving the tethered nucleic acid molecules tethered to said porous support.

2. The method of claim 1, wherein the enzymatically attaching adds an alkyne or azide group to the nucleic acid molecules.

3. The method of claim 1, wherein said enzymatically attaching comprises ligating an oligonucleotide comprising said reactive group to said nucleic acid molecules.

4. The method of claim 1, wherein the reactive sites on said porous support are alkyne or azide groups.

5. The method of claim 1, wherein the porous support is a silica membrane or packed beads containing reactive sites.

6. The method of claim 1, wherein the porous support is functionalized to contain reactive sites.

7. The method of claim 1, wherein the primer extension reaction uses gene specific primers that hybridize to a subset of the nucleic acid molecules that are tethered to the porous support.

8. The method of claim 1, wherein the primer extension reaction uses universal primers that hybridize to an oligonucleotide that is appended to said nucleic acid molecules.

9. The method of claim 1, wherein the sample comprises genomic DNA or cDNA that has been fragmented by physical, enzymatic or chemical treatment.

10. The method of claim 1, wherein the sample is obtained from a tissue biopsy.

11. The method of claim 1, wherein the sample is obtained from a patient having an infectious disease or condition.

12. The method of claim 1, wherein said eluting is done by:

treating the product of step c) with heat or a chaotrophic agent to denature the primer extension products from the tethered nucleic acid molecules; and

applying a force that separates the primer extension products and the porous support.

13. The method of claim 12, wherein the force is a centrifugal force.

14. The method of claim 1, further comprising analyzing the eluted primer extension products.

15. The method of claim 13, wherein the analyzing comprises sequencing the eluted primer extension products.

16. The method of claim 1, further comprising, after step d), performing a second primer extension reaction using said tethered nucleic acid molecules as a template to produce second primer extension products.

17. A kit comprising:

a porous support that comprises surface-exposed reactive sites; and

an oligonucleotide comprising a reactive group;

wherein said reactive sites and said reactive group are chosen such that they can specifically react with one another to produce a covalent linkage between the porous support and the oligonucleotide.

18. The kit of claim 17, wherein the porous support is composed of a silica membrane or packed beads comprising surface exposed azide or alkyne groups.

19. The kit of claim 17, wherein the kit comprises:

a spin column comprising;

a) a housing comprising an inlet and an outlet;

b) within said housing, said porous support; and

c) a first frit and a second frit, wherein said frits are porous an retain said porous support within said housing.

20. The kit of claim 17, wherein the housing contains lip that is dimensioned to fit over the wall of a microcentrifuge tube.