Sample preparation

Patent metadata

Field	Value
Publication number	US-10745740-B2
Application number	US-201313898064-A
Country	US
Kind code	B2
Filing date	May 20, 2013
Priority date	Mar 19, 2008
Publication date	Aug 18, 2020
Grant date	Aug 18, 2020

Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.

First claim

We claim:

1. A method of enriching, comprising:
a) providing i) an aqueous mix comprising PCR reagents, ii) template, iii) first and second PCR primers, said first PCR primers immobilized on emulsion beads for emulsion PCR, said second PCR primers comprising biotin, iv) an oil/detergent mix, and v) enrichment beads, wherein said enrichment beads comprise streptavidin;
b) adding said template, said first and second primers, said aqueous mix and said oil/detergent mix together so as to create one or more microemulsions, said one or more microemulsions comprising an aqueous compartment, each compartment comprising on average less than one template molecule;
c) temperature cycling said one or more microemulsions so as to amplify at least some of said template on at least some of said emulsion beads so as to create amplified product comprising biotin; and
d) enriching for beads with amplified template among said emulsion beads by contacting with said enrichment beads, wherein said emulsion beads with amplified template bind to said enrichment beads.

2. The method of claim 1 , wherein said enrichment beads comprise streptavidin and said binding to said enrichment beads in step d) proceeds through biotin-streptavidin binding.

3. The method of claim 1 , wherein said enrichment beads are magnetic.

4. The method of claim 1 , further comprising breaking said one or more microemulsions after step c) and before step d).

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

6. The method of claim 5 , wherein said sheared DNA fragments are attached to a linker with two primer recognition sequences.

7. The method of claim 6 , wherein said first primers are complementary to one of said primer recognition sequences and said second primers are complementary to the other primer recognition sequence.

8. A method of enriching, comprising:
a) providing i) an aqueous mix comprising PCR reagents, ii) template, iii) first and second PCR primers, said first PCR primers immobilized on emulsion beads for emulsion PCR, said second PCR primers comprising biotin, iv) an oil/detergent mix, and v) enrichment beads, wherein said enrichment beads comprise streptavidin;
b) adding said template, said first and second primers, said aqueous mix and said oil/detergent mix together so as to create one or more microemulsions, said one or more microemulsions comprising an aqueous compartment, each compartment comprising on average less than one template molecule;
c) temperature cycling said one or more microemulsions so as to amplify at least some of said template on at least some of said emulsion beads so as to create amplified product comprising biotin;
d) breaking said one or more microemulsions; and
e) enriching for beads with amplified template among said emulsion beads by contacting with said enrichment beads, wherein said emulsion beads with amplified template bind to said enrichment beads.

9. The method of claim 8 , wherein said enrichment beads comprise streptavidin and said binding to said enrichment beads in step d) proceeds through biotin-streptavidin binding.

10. The method of claim 8 , wherein said enrichment beads are magnetic.

11. The method of claim 8 , wherein said template comprises sheared DNA fragments.

12. The method of claim 11 , wherein said sheared DNA fragments are attached to a linker with two primer recognition sequences.

13. The method of claim 12 , wherein said first primers are complementary to one of said primer recognition sequences and said second primers are complementary to the other primer recognition sequence.

Assignees

• Qiagen Sciences Llc

Inventors

• Gordon Steven
• Veatch Phillip A

Classifications

• C12Q1/6806 Primary

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

• C12Q1/6844

  Nucleic acid amplification reactions · 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

• C12Q2537/159

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

• C12Q2563/137

  Metal/ion, e.g. metal label · CPC title