Sequencing reactions with monovalent cations for pulse width control

• US 10,184,148 B2
• Filed: 04/05/2017
• Issued: 01/22/2019
• 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, Lyle, John, Bjornson, Keith, Hanes, Jeremiah
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/480,226
• Publication Number:
  US 20170260579A1
• Time in Patent Office: 657 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 Na+ at a concentration of from about 1 mM to about 400 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 Na+ is selected such that the single molecule sequencing reaction has a median pulse width between about 10 msec and 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 about 300 bases.

4. The method of claim 1 wherein the concentration of Na+ is from about 5 mM to about 40 mM.

5. The method of claim 4 wherein the composition further comprises K+ at a concentration from about 50 mM to about 300 mM.

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

7. The method of claim 6 wherein the polymerase complex is immobilized onto the surface by attachment to the surface of the polymerase enzyme or the template nucleic acid.

8. The method of claim 6 wherein the polymerase enzyme complex is immobilized within a confined volume.

9. The method of claim 6 wherein the polymerase complex is immobilized in a zero mode waveguide.

10. The method of claim 1 wherein the plurality of nucleotide analogs comprise nucleic acids labeled on the phosphate portion of the nucleotide such that the label dissociates upon incorporation into the growing strand.

11. The method of claim 1 wherein the sequencing reaction mixture comprises a plurality of labeled nucleic acid analogs, and the nucleic acid analogs are labeled such that the label is cleaved upon incorporation of the nucleic acid analog into the growing strand, the observed pulses corresponding to the time period when the labeled nucleic acid is associated with the polymerase enzyme complex.

12. The method of claim 11 wherein the polymerase enzyme complex is immobilized onto a substrate.

13. The method of claim 12 wherein the polymerase enzyme complex is immobilized within a confined volume.

14. The method of claim 12 wherein the confined volume comprises a zero mode waveguide.

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

16. The method of claim 1 wherein the labeled nucleic acids comprise fluorescent or fluorogenic moieties.

17. The method of claim 1 wherein the labeled nucleic acids comprise fluorescent labels.