Signal confinement sequencing (SCS) and nucleotide analogues for signal confinement sequencing

• US 10,190,162 B2
• Filed: 10/23/2015
• Issued: 01/29/2019
• Est. Priority Date: 10/23/2014
• Status: Active Grant

• Current Assignee: MGI Tech Co., Ltd (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Snezana, Li, Handong, Drmanac, Radoje, Harness, Eric, Xu, Chongjun
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/921,466
• Publication Number:
  US 20160130647A1
• Time in Patent Office: 1,194 Days
• Field of Search: 435 61, 435 911, 536 41, 536 231
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

Novel fluorescent nucleotide analogs are provided herein. Also provided herein are methods of using the nucleotide analogs in sequencing-by-synthesis and signal confinement methods.

Claims

1. A method for sequencing a target nucleic acid, comprising:

a) providing a template nucleic acid immobilized on a surface, a primer, a polymerase, and a nucleotide analogue of the following structure;

2. The method of claim 1, wherein the binding molecule comprises an oligonucleotide.

3. The method of claim 1, wherein the capture element comprises a thio or thiol containing molecule.

4. The method of claim 1, wherein the capture element comprises streptavidin, an antibody, a protein, or a dendrimer.

5. The method of claim 1, wherein the capture element comprises an oligonucleotide immobilized complementary to the template nucleic acid.

6. The method of claim 1, wherein the template nucleic acid is an immobilized DNA concatemer comprising multiple copies of an adaptor sequence.

7. The method of claim 1, wherein L is a linking group and the linking group further comprises a quencher.

8. The method of claim 7, wherein the providing step comprises displacing the quencher from the linking group.

9. The method of claim 1, further comprising removing the fragment from the capture element on the surface.

10. The method of claim 9, wherein the removing step comprises heating the fragment and the capture element.

11. The method of claim 9, wherein the removing step comprises washing the fragment and the capture element with a buffer.

12. The method of claim 9, wherein the removing step comprises adding an enzyme to cleave the fragment from the capture element.

13. The method of claim 1, further comprising cleaving the blocking group from the incorporated nucleotide analogue.

14. The method of claim 1, wherein the detecting step is performed using fluorescence resonance energy transfer.

15. The method of claim 1, wherein L is absent.

16. The method of claim 1, wherein L is a linking group and the linking group is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted aryl.

17. The method of claim 16, wherein the linking group is a methylene group or a phenyl group.

18. The method of claim 1, wherein the binding molecule and capture element are a binding pair selected from biotin-streptavidin, biotin-avidin, antibody-antigen, and a complementary oligonucleotide pair capable of forming a nucleic acid duplexes.

19. The method of claim 1, wherein the blocking group is an amino-containing compound, an allyl-containing compound, an azido-containing compound, an alkoxy-containing compound, polyethylene glycol, or a substituted or unsubstituted alkyl.

20. The method of claim 1, wherein the blocking group is NH₂, —

CH₂CH═

CH₂, —

CH₂N₃, —

CH₂OCH₃, or polyethylene glycol.