METHODS FOR INCREASING ACCURACY OF NUCLEIC ACID SEQUENCING
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.
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) repeating steps a) and b), thereby determining a nucleotide sequence;
d) removing the template from the primer of step c);
e) adding a polynucleotide to a 3′ terminus of the primer of step d) to form a template;
f) exposing the template of step e) to a primer capable of hybridizing to said added polynucleotide to form template/primer duplex, and repeating steps a) through c) to sequence a portion of the template, wherein a least a portion of the sequence obtained is complementary to the nucleotide sequence of c), 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.