Loading extended polymerase-nucleic acid complexes

Patent metadata

Field	Value
Publication number	US-10934585-B2
Application number	US-201816007743-A
Country	US
Kind code	B2
Filing date	Jun 13, 2018
Priority date	Mar 23, 2011
Publication date	Mar 2, 2021
Grant date	Mar 2, 2021

Abstract

Compositions, methods and systems are provided for the loading of extended polymerase-nucleic acid complexes onto substrates. Primed polymerase-template complex comprising a polymerase enzyme and a circular nucleic acid template comprising a double stranded region connected at each end by a single-stranded hairpin region are provides in which the circular nucleic acid template comprises at least one reversible pause point. Nucleic acid synthesis is carried out such that a nascent strand is synthesized up to the reversible stop point producing an extended polymerase-template complex. The extended polymerase-template complex is then attached to a substrate. Such attached complexes can be used for single molecule sequencing in which the nucleic acid synthesis is re-initiated such that nucleic acid synthesis continues past the reversible stop point.

First claim

We claim:

1. A method for loading an extended polymerase-template complex onto a substrate comprising:
providing a primed polymerase-template complex comprising a polymerase enzyme and a circular nucleic acid template comprising a double stranded region connected at each end by a single-stranded hairpin region, wherein the circular nucleic acid template comprises a reversible stop point;
providing reagents for carrying out nucleic acid synthesis whereby the polymerase enzyme synthesizes a nascent strand up to the reversible stop point, thereby producing an extended polymerase-template complex; and
exposing the extended polymerase-template complex to a substrate, whereby the extended polymerase-template complex is attached to the substrate.

2. The method of claim 1 wherein the extended polymerase-template complex is attached to the substrate through the polymerase enzyme.

3. The method of claim 1 wherein the extended polymerase-template complex is attached to the substrate through the template.

4. The method of claim 1 wherein the substrate comprises nanoscale wells and the extended polymerase-template complex is attached within a nanoscale well on the substrate.

5. The method of claim 4 wherein the extended polymerase-template complex is attached to the base of the nanoscale well.

6. The method of claim 1 wherein the extended polymerase-template complex is attached within a zero mode waveguide on the substrate.

7. The method of claim 1 wherein the reversible stop point is within a single stranded hairpin region.

8. The method of claim 1 wherein the polymerase enzyme in the primed polymerase-enzyme complex is within a single stranded hairpin region.

9. The method of claim 1 wherein the polymerase enzyme in the primed polymerase-enzyme complex is within a single stranded hairpin region and the reversible stop point is within a single stranded hairpin region.

10. The method of claim 8 wherein the reversible stop point is in the same hairpin as the hairpin with the primed polymerase enzyme complex.

11. The method of claim 10 wherein the reversible stop point is upstream of the primed polymerase enzyme complex.

12. The method of claim 1 wherein the single stranded hairpin loops are identical.

13. The method of claim 1 wherein each end of the circular nucleic acid template comprises a different single stranded hairpin adaptor.

14. The method of claim 1 wherein the reversible stop point comprises a reversibly bound blocking group.

15. The method of claim 1 wherein the reversible stop point comprises a photolabile group.

16. The method of claim 1 wherein the reversible stop point comprises a non-native nucleotide.

17. The method of claim 1 wherein the reversible stop point comprises sequence recognized by a binding protein.

18. The method of claim 17 wherein the binding protein comprises a transcription factor.

19. The method of claim 17 wherein the binding protein comprises a lac repressor protein.

20. The method of claim 1 wherein the reversible stop point comprises a fifth base.

21. The method of claim 1 wherein the reversible stop point comprises a damaged base that can be repaired by the addition of a repair enzyme.

22. The method of claim 1 wherein the reversible stop point comprises an abasic site.

23. The method of claim 1 wherein the reversible stop point comprises a nick in the template nucleic acid.

24. The method of claim 1 , further comprising carrying out single molecule sequencing of the circular nucleic acid template by re-initiating nucleic acid synthesis by the polymerase enzyme such that the polymerase enzyme synthesized nucleic acid past the reversible stop point.

Assignees

• Pacific Biosciences California Inc

Inventors

• Pham Thang
• Bhamidipati Arunashree
• Travers Kevin
• Olivares Eric
• Clark Tyson A
• Korlach Jonas

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N33/54353

  with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393) · CPC title

• G01N33/54313

  the carrier being characterised by its particulate form · CPC title

• G01N2333/922

  Ribonucleases (RNAses); Deoxyribonucleases (DNAses) · CPC title

• C40B60/14

  for creating libraries · CPC title