Methods for increasing accuracy of nucleic acid sequencing

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.

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.