Sequencing reactions with lithium for pulse width control

• US 9,650,671 B2
• Filed: 03/09/2015
• Issued: 05/16/2017
• Est. Priority Date: 07/12/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Fedorov, Andrei, Bjornson, Keith, Hanes, Jeremiah, Lyle, John
• Primary Examiner(s): Strzelecka, Teresa
• Application Number:
  US14/642,307
• Publication Number:
  US 20150203908A1
• Time in Patent Office: 799 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label

Abstract

Compositions, kits, methods and systems for single molecule nucleotide sequencing comprising producing polymerase reactions having lithium that control the median pulse width for incorporated nucleotides are disclosed. The levels of lithium are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.

Claims

1. A method for performing single molecule sequencing having a pulse width within a desired range comprising:

providing a composition comprising;

a modified recombinant phi-29 type DNA polymerase enzyme having at least 80% sequence similarity to SEQ ID NO;

1, a primer, a template nucleic acid, and a plurality of labeled nucleotide analogs, wherein the composition comprises Li+ at a concentration of from about 0.05 mM to about 40 mM; and

observing the association of the labeled nucleotide analogs with a complex of the polymerase, primer, and template nucleic acid over time to determine a sequence of the template nucleic acid, wherein the concentration of Li+ is selected such that the single molecule sequencing reaction has a median pulse width from about 10 msec to about 400 msec.

2. The method of claim 1 wherein the single molecule sequencing reaction has a median interpulse distance from about 300 msec to about 1.5 s.

3. The method of claim 1 wherein the single molecule sequencing reaction has a median read length greater than 300 bases.

4. The method of claim 1 wherein the concentration of Li+ is from about 0.1 mM to about 4 mM.

5. The method of claim 1 wherein the composition further comprises K+ at a concentration from about 100 mM to about 400 mM.

6. The method of claim 5 wherein the concentration of K+ is from about 150 mM to about 250 mM.

7. The method of claim 1 wherein the polymerase enzyme, primer, and template nucleic acid comprise a polymerase complex that is immobilized on a surface.

8. The method of claim 7 wherein the polymerase complex is immobilized onto the surface by attachment to the surface of the polymerase enzyme or the template nucleic acid.

9. The method of claim 7 wherein the polymerase enzyme complex is immobilized within a confined volume.

10. The method of claim 7 wherein the polymerase complex is immobilized in a zero mode waveguide.

11. The composition of claim 1 wherein the plurality of nucleotide analogs are labeled on the phosphate portion such that the label dissociates upon incorporation into the growing strand.

12. The method of claim 1 wherein the plurality of labeled nucleotide analogs are labeled such that the label is cleaved upon incorporation of the nucleotide analog into the growing strand, the observed pulses corresponding to the time period when the labeled nucleotide analog is associated with the polymerase enzyme complex.

13. The method of claim 12 wherein the polymerase enzyme complex is immobilized onto a substrate.

14. The method of claim 13 wherein the polymerase enzyme complex is immobilized within a confined volume.

15. The method of claim 13 wherein the confined volume comprises a zero mode waveguide.

16. The method of claim 1 wherein the observing is carried out using optical detection.

17. The method of claim 1 wherein the plurality of labeled nucleotide analogs comprise fluorescent or fluorogenic moieties.

18. The method of claim 1 wherein the plurality of labeled nucleotide analogs comprise fluorescent labels.