Apparatus and method for performing nucleic acid analysis

Abstract

The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.

1 . A method of increasing accuracy of nucleic acid sequencing, the method comprising the steps of:
 a) performing a first single molecule sequencing reaction on at least a first region of a template, thereby obtaining a nucleotide sequence of at least the first region of the template; and   b) performing a second single molecule sequencing reaction on at least the first region of the template to resequence the first region of the template, thereby increasing the accuracy of nucleic acid sequencing.   
     
     
         2 . The method of  claim 1 , wherein the sequence obtained in a) is compared with the sequence obtained in b). 
     
     
         3 . The method of  claim 1 , further comprising the step of performing at least a third single molecule sequencing reaction on at least the first region of the template. 
     
     
         4 . The method of  claim 1 , wherein:
 the first single molecule sequencing reaction comprises exposing a template/first primer duplex to a polymerase and one or more nucleotides comprising a detectable label under conditions sufficient for template-dependent nucleotide addition to the first primer, the first primer being hybridized to the first region of the template, wherein the template/first primer duplex is individually optically resolvable.   
     
     
         5 . The method of  claim 4 , wherein the second single molecule sequencing reaction comprises continuing the template-dependent nucleotide addition to the first primer. 
     
     
         6 . The method of  claim 4 , wherein the label is an optically-detectable label. 
     
     
         7 . The method of  claim 6 , wherein the optically-detectable label is a fluorescent label. 
     
     
         8 . The method of  claim 7 , wherein the fluorescent label is selected from the group consisting of fluorescein, rhodamine, cyanine, Cy5, Cy3, BODIPY, alexa, and derivatives thereof. 
     
     
         9 . The method of  claim 4 , wherein the template/first primer duplex is attached to a surface. 
     
     
         10 . The method of  claim 1 , wherein a plurality of regions are sequenced. 
     
     
         11 . The method of  claim 10 , wherein a plurality of regions are resequenced. 
     
     
         12 . A method of increasing accuracy of nucleic acid sequencing, the method comprising the steps of:
 a) performing a first set of single molecule sequencing reactions on a plurality of regions of a template, thereby obtaining a nucleotide sequence of at least one of the plurality of regions of the template; and   b) performing a second set of single molecule sequencing reactions on the plurality of regions of the template to resequence the at least one of the plurality of regions of the template, thereby increasing the accuracy of nucleic acid sequencing.   
     
     
         13 . The method of  claim 12 , wherein the sequence obtained in a) is compared with the sequence obtained in b). 
     
     
         14 . The method of  claim 12 , further comprising the step of performing at least a third set of single molecule sequencing reactions on the at least one of the plurality of regions of the template. 
     
     
         15 . The method of  claim 12 , wherein the sequencing reactions comprise exposing a duplex comprising the template and at least one primer to a polymerase and to one or more nucleotides comprising a detectable label under conditions for template-dependent nucleotide addition to the primer, which is hybridized to the template, wherein the detectable label is an optically-detectable label, and further wherein the duplex is individually optically resolvable. 
     
     
         16 . The method of  claim 15 , wherein the optically-detectable label is a fluorescent label. 
     
     
         17 . The method of  claim 16 , wherein the fluorescent label is selected from the group consisting of fluorescein, rhodamine, cyanine, Cy5, Cy3, BODIPY, alexa, and derivatives thereof. 
     
     
         18 . The method of  claim 17 , wherein the duplex is attached to a surface. 
     
     
         19 . The method of  claim 12 , wherein the template is a circular template. 
     
     
         20 . The method of  claim 12 , wherein the template is a concatemeric template.