Purified extended polymerase/template complex for sequencing

• US 8,936,911 B2
• Filed: 09/15/2011
• Issued: 01/20/2015
• Est. Priority Date: 09/22/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Sun, Lei, Park, Insil
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US13/233,585
• Publication Number:
  US 20120071359A1
• Time in Patent Office: 1,223 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Methods, Compositions, and Systems are provided for obtaining polymerase-template complex mixtures with improved levels of active polymerase. In some aspects, methods are described in which a polymerase-template complex is exposed to reaction conditions in which a complementary strand to the template is produced. The extended reaction mixture is purified, for example by gel filtration chromatography to produce a mixture of polymerase-template complex having a higher active fraction. This purified mixture can be used for further analyses including single molecule sequencing.

Claims

1. A method comprising:

producing a reaction mixture comprising a polymerase enzyme complex having a polymerase enzyme and a circular template nucleic acid, the reaction mixture comprising reagents for template directed nucleic acid synthesis including nucleotides, nucleotide analogs or both;

extending a complementary strand such that a portion of the polymerase enzyme complexes have complementary strands within a desired size range;

purifying the reaction mixture based on the size of the complexes to enhance the relative amount of polymerase enzyme complex having complementary strands within the desired size range;

loading the purified reaction mixture onto a substrate.

2. The method of claim 1 wherein a component of the reaction mixture is provided in a limiting amount that causes the extending of the complementary strand to halt when a portion of the polymerase enzyme complexes have complementary strands within the desired size range.

3. The method of claim 2 wherein the component provided in a limiting amount is one or more of the nucleotides or nucleotide analogs.

4. The method of claim 1 wherein the reaction is halted at a time selected such that a portion of the polymerase enzyme complexes have complementary strands within a desired size range.

5. The method of claim 4 wherein the reaction is halted using a metal salt.

6. The method of claim 5 wherein the metal salt comprises Ca++.

7. The method of claim 1 wherein the template nucleic acid comprises a single circular strand of DNA having a self-complementary region, and having hairpin regions at the two ends of the complementary region.

8. The method of claim 1 wherein the template nucleic acid comprises sequences from a nucleic acid library.

9. The method of claim 1 wherein the complex also comprises a primer hybridized to the nucleic acid template.

10. The method of claim 1 wherein the desired size range of the complementary strand is from 100 bases to 100K bases.

11. The method of claim 1 wherein purifying the reaction mixture comprises the use of size exclusion chromatography, gel permeation, or gel filtration chromatography.

12. The method of claim 1 wherein the polymerase enzyme complex is stabilized during purification with Ca++.

13. The method of claim 1 wherein the polymerase enzyme complex is stabilized during purification with one or more nucleotides or nucleotide analogs.

14. The method of claim 1 wherein the polymerase enzyme complex is stabilized during purification with Ca++ and one or more nucleotides or nucleotide analogs.

15. The method of claim 1 wherein the polymerase enzyme comprises a DNA polymerase.

16. The method of claim 15 wherein the DNA polymerase comprises a phi-29 type polymerase.

17. The method of claim 1 wherein the polymerase enzyme comprises a reactive element for attachment to the substrate.

18. The method of claim 1 wherein the substrate onto which the purified reaction mixture is loaded comprises an array of reactive sites that react with the polymerase to immobilize the polymerase enzyme complex onto the substrate.

19. The method of claim 1 wherein the substrate onto which the purified reaction mixture is loaded comprises an array of nanoscale wells.