Sequencing reactions with alkali metal cations for pulse width control

• US 8,986,930 B2
• Filed: 07/07/2011
• Issued: 03/24/2015
• 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:
  US13/177,775
• Publication Number:
  US 20120009567A1
• Time in Patent Office: 1,356 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 alkali metals such as lithium, sodium, potassium, rubidium, and cesium in the polymerization are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.

Claims

1. A composition for single molecule nucleic acid sequencing 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 a mixture of alkali metals comprising K+ at a concentration of from about 50 mM to about 300 mM, and Li+ at a concentration of from about 0.05 mM and about 40 mM.

2. The composition of claim 1 wherein the single molecule sequencing reaction has a median pulse width between about 10 msec and about 400 msec.

3. The composition of claim 1 wherein the single molecule sequencing reaction has an interpulse distance is from about 300 msec to about 1.5 s.

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

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

6. The composition of claim 1 wherein the concentration of K+ is from about 100 mM to about 300 mM.

7. A method for single molecule sequencing comprising:

providing the composition of claim 1,

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 levels of K+ and Li+ are selected such that the single molecule sequencing reaction has a median pulse width between about 10 msec and about 400 msec.

8. The method of claim 7 wherein the single molecule sequencing reaction has an interpulse distance is from about 300 msec to about 1.5 s.

9. The method of claim 7 wherein the single molecule sequencing reaction has a median read length greater than about 300 bases.

10. The method of claim 7 wherein the concentration of Li+ is from about 0.1 mM to about 4 mM.

11. The method of claim 7 wherein the concentration of K+ is from about 100 mM to about 300 mM.

12. The composition of claim 1 wherein the modified recombinant phi-29 DNA polymerase enzyme has at least 90% sequence similarity to SEQ ID NO:

1.