Nucleic acid synthesis compositions and methods and systems for using same

• US 8,658,365 B2
• Filed: 08/06/2009
• Issued: 02/25/2014
• Est. Priority Date: 03/31/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bjornson, Keith, Lee, Harold Ray, Korlach, Jonas
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US12/537,130
• Publication Number:
  US 20100047802A1
• Time in Patent Office: 1,664 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

The Application relates to compositions, kits, methods, and systems for nucleotide sequencing comprising producing polymerase reactions that comprise both catalytic and non-catalytic divalent metal ions. Effective ratios and amounts of catalytic and non-catalytic divalent metal ions are described.

Claims

1. A method of sequencing a nucleic acid, comprising:

providing a complex comprising a Phi-29 DNA polymerase enzyme, a template nucleic acid, and primer sequence complementary to at least a portion of the template nucleic acid;

contacting the complex with a reaction mixture that comprises a mixture of divalent metal ions comprising at least one catalytic metal ion and at least one non-catalytic metal ion, wherein the catalytic metal is selected from Mg²⁺, Mn²⁺ and mixtures thereof, and the non-catalytic metal is selected from Ca²⁺, Sr²⁺, or Ba²⁺ and mixtures thereof, and one or more types of nucleotides or nucleotide analogs wherein a ratio of catalytic metal to non-catalytic metal in the reaction mixture is from about 10;

1 to about 1;

5;

detecting incorporation of one or more of the nucleotide into the complex; and

using the detected incorporation to determine the sequence of the template nucleic acid.

2. The method of claim 1, wherein the catalytic metal comprises Mn²⁺ and the non-catalytic metal comprises Ca²⁺.

3. The method of claim 1, wherein a ratio of catalytic metal to non-catalytic metal in the reaction mixture is from about 5:

1 to about 1;

1.

4. The method of claim 1, wherein a ratio of catalytic metal to non-catalytic metal in the reaction mixture is from about 2.5:

1 to about 1.5;

1.

5. The method of claim 1, wherein the catalytic metal and non-catalytic metal are present in the reaction mixture at a total concentration of from about 0.1 mM to about 10 mM.

6. The method of claim 1, wherein the detecting step comprises detecting incorporation of nucleotides in real-time as they are incorporated into the complex.

7. The method of claim 6, wherein the complex is immobilized on a solid support.

8. The method of claim 7, wherein the complex is immobilized on a solid support in an individually optically resolvable configuration.

9. A method of sequencing a nucleic acid, comprising:

providing a complex comprising a Phi-29 DNA polymerase enzyme, a template nucleic acid, and a primer sequence complementary to at least a portion of the template nucleic acid;

contacting the complex with a reaction mixture that comprises a plurality of types of nucleotides, and a mixture of divalent metal ions comprising at least one catalytic metal ion and at least one non-catalytic metal ion at first and second concentrations, respectively, wherein the catalytic metal is selected from Mg2+, Mn2+ and mixtures thereof, and the non-catalytic metal is selected from Ca2+, Sr2+, or Ba2+ and mixtures thereof wherein a ratio of catalytic metal to non-catalytic metal in the reaction mixture is from about 10;

1 to about 1;

5, wherein the mixture of divalent metal ions is selected to provide improved sequencing accuracy over a complex exposed to the first concentration of the catalytic metal in the absence of the non-catalytic metal;

detecting incorporation of a nucleotide into the complex; and

using the detected incorporation to determine the sequence of the template nucleic acid.

10. The method of claim 9 wherein the Phi-29 DNA polymerase enzyme comprises a modified recombinant Phi-29 DNA polymerase enzyme.