Method for sequencing a polynucelotide template

• US 9,994,896 B2
• Filed: 12/17/2014
• Issued: 06/12/2018
• Est. Priority Date: 02/08/2006
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Smith, Geoffrey Paul, Boutell, Jonathan Mark, Barnes, Colin Lloyd, Rigatti, Roberto, Gormley, Niall Anthony, Bentley, David, Ost, Tobias William Barr, Smith, Vincent Peter, Worsley, Graham John, Vermaas, Eric Hans
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US14/574,023
• Publication Number:
  US 20150203910A1
• Time in Patent Office: 1,273 Days
• Field of Search: None
• 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 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention provides methods for pairwise sequencing of a double-stranded polynucleotide template, which methods result in the sequential determination of nucleotide sequences in two distinct and separate regions of the polynucleotide template.

Claims

1. A method for sequencing, the method comprising:

(a) amplifying a template to generate a single-stranded polynucleotide, wherein the single-stranded polynucleotide comprises;

a first region of known sequence;

a first template region;

a second region of known sequence, wherein the first template region is between the first region of known sequence and the second region of known sequence;

a second template region; and

a third region of known sequence, wherein the second template region is between the second region of known sequence and the third region of known sequence;

(b) disposing the single-stranded polynucleotide on a planar support to generate a single-stranded polynucleotide immobilized on the planar support, wherein the single-stranded polynucleotide forms part of a colony comprising identical copies of the single-stranded polynucleotide, the solid support comprising an array of colonies located at discrete sites on the solid support;

(c) hybridizing a first primer to the first region of known sequence in the single-stranded polynucleotide immobilized on the planar support;

(d) extending the first primer to form a first extended primer hybridized to the single-stranded polynucleotide, wherein the first extended primer comprises a fluorescent label;

(e) detecting fluorescence emitted from the fluorescent label in the first extended primer while the first extended primer is hybridized to the single-stranded polynucleotide immobilized on the planar support;

(f) denaturing the first extended primer from the single-stranded polynucleotide;

(g) hybridizing a second primer to the second region of known sequence in the single-stranded polynucleotide immobilized on the planar support, wherein the second primer is hybridized to the single-stranded polynucleotide after denaturing the first extended primer from the single-stranded polynucleotide;

(h) extending the second primer to form a second extended primer hybridized to the single-stranded polynucleotide immobilized on the planar support, wherein the second extended primer comprises a fluorescent label; and

(i) detecting fluorescence emitted from the fluorescent label in the second extended primer while the second extended primer is hybridized to the single-stranded polynucleotide.

2. The method of claim 1, wherein extending the first primer comprises extending the first primer in the 5′

to 3′

direction.

3. The method of claim 1, wherein extending the second primer comprises extending the second primer in the 5′

to 3′

direction.

4. The method of claim 1, wherein the solid support comprises silicon.

5. The method of claim 1, wherein denaturing the first extended primer comprises chemical denaturing.

6. The method of claim 1, wherein denaturing the first extended primer comprises heating.

7. The method of claim 1, wherein the template comprises a circular template.

8. The method of claim 7, wherein amplifying comprises rolling circle amplification.

9. The method of claim 1, wherein extending the first primer comprises extending the first primer using a ligase.

10. The method of claim 1, wherein extending the second primer comprises extending the second primer using a ligase.

11. The method of claim 1, wherein the first region of known sequence comprises a recognition site for a restriction enzyme.

12. The method of claim 11, wherein the restriction enzyme is mmeI or EcoP15.

13. The method of claim 1, wherein the third region comprises a recognition site for a restriction enzyme.

14. The method of claim 13, wherein the restriction enzyme is mmeI or EcoP15.

15. The method of claim 1, wherein the single-stranded polynucleotide is non-covalently immobilized on the planar support.

16. The method of claim 1, wherein the single-stranded polynucleotide is directly immobilized on the planar support.