Methods for increasing accuracy of nucleic acid sequencing

• US 20070243535A1
• Filed: 04/14/2006
• Published: 10/18/2007
• Est. Priority Date: 04/14/2006
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Harris, Timothy
• Granted Patent: US 7,282,337 B1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The invention provides methods for improving the accuracy of a sequencing-by-synthesis reaction by sequencing at least a portion of a template and at least a portion of template complementary sequence.

Claims

1. A method of increasing accuracy of nucleic acid sequencing, the method comprising the steps of:

a) exposing a duplex comprising a template and a primer to a polymerase and one or more nucleotide comprising a detectable label under conditions sufficient for template-dependent nucleotide addition to said primer, wherein said template is individually optically resolvable;

b) identifying nucleotide incorporated into said primer;

c) neutralizing said detectable label or removing said detectable label from said incorporated nucleotide;

d) repeating steps a)-c), thereby determining a nucleotide sequence incorporated into said primer of said duplex;

e) removing the template from the primer of step d);

f) adding a polynucleotide to a 3′

terminus of the primer from step e) to form a template;

g) exposing the template of step f) to a primer capable of hybridizing to said added polynucleotide to form template/primer duplex, and repeating steps a) through d) to sequence a portion of the template, wherein at least a portion of the sequence obtained is complementary to the nucleotide sequence of d), thereby increasing the accuracy of nucleic acid sequencing.

2. The method of claim 1, wherein the sequence obtained in c) is compared with a complement of the sequence obtained in f).

3. The method of claim 1, further comprising repeating steps d) and f).

4. The method of claim 1, wherein said label is an optically-detectable label.

5. The method of claim 4, wherein said optically-detectable label is a fluorescent label.

6. The method of claim 5, wherein said fluorescent label is selected from the group consisting of fluorescein, rhodamine, cyanine, Cy5, Cy3, BODIPY, alexa, and derivatives thereof.

7. The method of claim 1, wherein said duplex is attached to a surface.

8. The method of claim 1, wherein said primer is attached to a surface.