Analyte enrichment methods and compositions

Patent metadata

Field	Value
Publication number	US-10626446-B2
Application number	US-201816178262-A
Country	US
Kind code	B2
Filing date	Nov 1, 2018
Priority date	May 13, 2013
Publication date	Apr 21, 2020
Grant date	Apr 21, 2020

Abstract

Methods and compositions for enriching a population of particles containing an analyte are disclosed. In one embodiment, enrichment beads are used that are larger in size than the beads used for amplification. A separation device is employed that can retain larger beads with bound amplified beads. The technique finds many uses, including enriching for beads with clonally amplified template, which can be used in a variety of assays, including nucleic acid sequencing.

First claim

The invention claimed is:

1. A method of enriching, comprising:
a) providing
i) an emulsion comprising one or more aqueous compartments in oil, at least some of said compartments comprising PCR reagents, a first PCR primer immobilized on an emulsion bead, a second biotinylated PCR primer in solution, and a nucleotide sequence template; and
ii) enrichment beads, wherein said enrichment beads are larger than emulsion beads in said compartments;

b) exposing said emulsion to conditions so as to amplify at least some of said template to produce amplified template on at least some of said emulsion beads in at least some of said compartments, wherein one strand of said amplified template terminates with biotin; and
c) enriching for emulsion beads comprising said amplified template by contacting said emulsion beads with said enrichment beads, wherein said emulsion beads comprising said amplified template bind to said enrichment beads so as to make a population of emulsion bead—enrichment bead complexes; and
d) capturing at least some of said population of complexes under conditions such that a majority of said emulsion beads not comprising amplified template are not captured.

2. The method of claim 1 , further comprising, after step d):
e) subjecting said population of complexes to conditions so as to separate said emulsion beads comprising amplified template from said enrichment beads such that the majority of said emulsion beads comprising amplified template separate from said enrichment beads.

3. The method of claim 2 , wherein said emulsion beads comprising amplified template are separated from said enrichment beads by centrifugation.

4. The method of claim 2 , wherein said conditions of step e) comprise denaturing conditions.

5. The method of claim 2 , wherein said emulsion beads separated from said enrichment beads are magnetic.

6. The method of claim 5 , wherein said emulsion beads separated from said enrichment beads are exposed to a magnet.

7. The method of claim 2 , further comprising recovering said separated emulsion beads comprising amplified template so as to create an enriched population contaminated with less than 10% of beads without amplified template.

8. The method of claim 7 , wherein said enriched population is contaminated with less than 1% of beads without amplified template.

9. The method of claim 7 , wherein said enriched population comprises between 1 and 20 million beads.

10. The method of claim 1 , wherein each compartment comprises on average less than one template.

11. The method of claim 1 , further comprising breaking said emulsion after step b) and before step c).

12. The method of claim 1 , wherein said enrichment beads comprise streptavidin.

13. The method of claim 1 , wherein the capturing in step d) comprises size selection.

14. The method of claim 13 , wherein said size selection comprises density centrifugation.

15. The method of claim 13 , wherein said size selection comprises capturing at least some of said population of complexes on a surface.

16. The method of claim 15 , wherein said surface comprises the surface of a filter.

17. The method of claim 16 , wherein said filter is a single layer nylon mesh.

18. The method of claim 16 , wherein said filter is positioned in a spin column.

19. The method of claim 18 , wherein said spin column is centrifuged during step d) so as to facilitate passage of said uncaptured emulsion beads through said filter.

20. The method of claim 1 , wherein at least a portion of said enrichment beads binds more than one of said emulsion beads comprising amplified template.

21. The method of claim 1 , wherein said PCR reagents comprise a polymerase and nucleotides or nucleotide analogues in a buffer.

22. The method of claim 1 , wherein said template comprises sheared DNA fragments.

23. The method of claim 22 , wherein said sheared DNA fragments comprise 3′ and 5′ adaptors.

24. The method of claim 23 , wherein said first PCR primer and said second PCR primer are complementary to a portion of one of said adaptors.

25. The method of claim 1 , wherein said exposing to conditions of step b) comprises temperature cycling.

26. The method of claim 1 , wherein said emulsion beads in said compartments are magnetic and said magnetic beads are recovered after step b), and thereafter exposed to a magnet, and washed.

27. The method of claim 1 , wherein said emulsion beads are 1 μm in diameter and said enrichment beads are approximately 15 μm in diameter.

Assignees

• Qiagen Sciences Llc

Inventors

• Olejnik Jerzy
• Gordon Steven
• Werner Martina

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6806 Primary

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

• C12Q2563/159

  Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components · CPC title

• C12Q2563/131

  the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin · CPC title

• C12Q2537/159

  Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions · CPC title