Sequencing reactions with monovalent cations for pulse width control

• US 10,612,087 B2
• Filed: 12/12/2018
• Issued: 04/07/2020
• 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:
  US16/218,280
• Publication Number:
  US 20190194741A1
• Time in Patent Office: 482 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 monovalent cations that control the median pulse width for incorporated nucleotides are disclosed. The levels of monovalent cations are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.

Claims

1. A composition for a single molecule nucleic acid sequencing reaction 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 K⁺ at a concentration from about 50 mM to about 300 mM.

2. The composition of claim 1, wherein the concentration of Na⁺ is from about 5 mM to about 40 mM.

3. The composition of claim 1, wherein the polymerase enzyme, primer, and template nucleic acid are in the form of a polymerase complex.

4. The composition of claim 3, wherein the polymerase complex is immobilized onto a surface.

5. The composition of claim 4, wherein the polymerase complex is immobilized onto the surface by attachment of the polymerase enzyme to the surface.

6. The composition of claim 4, wherein the polymerase complex is immobilized onto the surface by attachment of the primer to the surface.

7. The composition of claim 4, wherein the polymerase complex is immobilized onto the surface by attachment of the template nucleic acid to the surface.

8. The composition of claim 4, wherein the polymerase complex is immobilized onto the surface within a confined volume.

9. The composition of claim 8, wherein the polymerase complex is immobilized onto the surface of a zero mode waveguide.

10. The composition of claim 1, wherein each of the plurality of labeled nucleotide analogs comprises a label on a polyphosphate portion of the nucleotide analog.

11. The composition of claim 10, wherein the label on each of the plurality of labeled nucleotide analogs comprises a fluorescent or fluorogenic moiety.

12. The composition of claim 11, wherein the label on each of the plurality of labeled nucleotide analogs comprises a fluorescent moiety.

13. The composition of claim 12, wherein the plurality of labeled nucleotide analogs consists of A, G, T, and C nucleotide analogs, wherein each of the A, G, T, and C nucleotide analogs is labeled with a different fluorescent dye.