Granted Patent B1

US 7,282,337 · App. 11/404,695 · Granted Oct 16, 2007

Abstract

Claims (15)

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 (e) through (g).

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.

Assignments (8)