Asynchronous sequencing of biological polymers

• US 8,993,230 B2
• Filed: 12/04/2008
• Issued: 03/31/2015
• Est. Priority Date: 12/04/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Peluso, Paul S., Eid, John S., Rank, David
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US12/328,715
• Publication Number:
  US 20100143900A1
• Time in Patent Office: 2,308 Days
• Field of Search: 435/6, 435/91.2, 536/24.33
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

The present invention provides methods, reagents and apparatus for conducting single molecule sequencing in an asynchronous manner. The subject methods and compositions are particularly useful for high throughput and multiplexed sequencing.

Claims

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

(a) providing a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule comprising the same sequence as the target nucleic acid molecule, wherein the first nucleic acid molecule is complexed with a first primer and a first polymerase and the second nucleic acid molecule is complexed with a second primer and a second polymerase, wherein the first polymerase has a different rate of polymerization from the second polymerase, and further wherein the first nucleic acid molecule is a first circular template and the second nucleic acid molecule is a second circular template;

(b) subjecting the first circular template to a first polymerization reaction to yield a first strand complementary to the first nucleic acid molecule;

(c) subjecting the second circular template to a second polymerization reaction to yield a second strand complementary to the second nucleic acid molecule, wherein the first polymerization reaction is asynchronous compared to the second polymerization reaction, the asynchronous due to different rates of polymerization exhibited by the first polymerase on the first circular template and the second polymerase on the second circular template;

(d) obtaining overlapping time sequences of incorporation of nucleotides or nucleotide analogs into the first strand and the second strand during the asynchronous polymerization reactions; and

(e) sequencing the target nucleic acid molecule by compiling, using a programmed computer, the overlapping time sequences of incorporation of nucleotides into the first and second strands obtained during the asynchronous polymerization reactions.

2. The method of claim 1, wherein each of the first strand and second strand shares sequence identity to a contiguous segment of the target nucleic acid molecule having a length that is no more than about 95% of the entire length of the target nucleic acid molecule.

3. The method of claim 1, wherein each of the first strand and second strand shares sequence identity to a contiguous segment of the target nucleic acid molecule having a length that is no more than about 75% of the entire length of the target nucleic acid molecule.

4. The method of claim 1, wherein each of the first strand and second strand shares sequence identity to a contiguous segment of the target nucleic acid molecule having a length that is no more than about 50% of the entire length of the target nucleic acid molecule.

5. The method of claim 1, wherein the first primer and the second primer have the same sequence of nucleotides or nucleotide analogs.

6. The method of claim 1, wherein the first primer and the second primer have different sequences of nucleotides or nucleotide analogs.

7. The method of claim 1, wherein the first circular template and the second circular template are attached to a support surface.

8. The method of claim 1, wherein the first polymerase and the second polymerase are DNA polymerases.

9. The method of claim 1, wherein the first polymerase and the second polymerase are polymerases exhibiting strand-displacement activity.

10. The method of claim 1, wherein the target nucleic acid molecule is selected from deoxyribonucleic acid, ribonucleic acid, locked nucleic acid, glycol nucleic acid and threose nucleic acid.

11. The method of claim 1, wherein the overlapping time sequences of incorporation of nucleotides or nucleotide analogs into the first strand and the second strand represent segments of the target nucleic acid sequence.

12. The method of claim 1, wherein the first strand and the second strand overlap by about 5 or more nucleotides.

13. The method of claim 1, wherein the first strand and the second strand overlap by about 10 or more nucleotides.

14. The method of claim 1, wherein the first strand and the second strand overlap by about 20 or more nucleotides.

16. The method of claim 1, wherein the first polymerase and the second polymerase are attached to a support surface.

15. A method for sequencing a target nucleic acid molecule, the method comprising:

(a) providing two or more circular nucleic acid molecules comprising the same sequences as the target nucleic acid molecule, wherein each of the two or more circular nucleic acid molecules is complexed with a primer and a polymerase, wherein individual polymerases are complexed at different locations on the circular nucleic acid molecules such that at least two nascent strands of different but overlapping sequences are achieved;

(b) subjecting the two or more circular nucleic acid molecules to polymerization reactions to yield the at least two nascent strands, wherein the polymerization reactions are asynchronous polymerization reactions due to the different locations of the individual polymerases on the circular nucleic acid molecules;

(c) obtaining overlapping time sequences of incorporation of nucleotides or nucleotide analogs into the nascent strands of different but overlapping sequences during the asynchronous polymerization reactions; and

(d) aligning and compiling the overlapping time sequences of incorporation obtained during the asynchronous polymerization reactions, thereby sequencing the target nucleic acid molecule.