Methods for sequencing a polynucleotide template

Abstract

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

1 . (canceled) 
     
     
         2 . A method for pairwise sequencing of a polynucleotide template, the method comprising:
 (a) providing a polynucleotide template immobilized on a surface, said polynucleotide template comprising a partially self-complementary covalently closed circle structure comprising
 a fully double-stranded, blunt ended template comprising a first template strand having a first region and a second template strand, 
 a first hairpin linker immobilized on the surface, wherein the 5′ end of the first hairpin linker is linked to the 3′ end of said first template strand and the 3′ end of the first hairpin linker is linked to the 5′ end of said second template strand, and 
 a second hairpin linker, wherein the 5′ end of the second hairpin linker is linked to the 3′ end of said second template strand and the 3′ end of the second hairpin linker is linked to the 5′ end of said first template strand; 
   (b) cleaving the polynucleotide template, thereby yielding a cleaved polynucleotide template comprising a 3′ end that comprises a first free 3′ hydroxyl group used for initiating sequencing of the first region of the first template strand, wherein the cleaving comprises contacting the polynucleotide template with a nicking endonuclease to generate a nick at a site before, at, or beyond the 3′ end of the first hairpin linker;   (c) performing a first sequencing-by-synthesis reaction comprising sequential incorporation of different complementary reversibly-terminated nucleotides into the 3′ end, thereby determining the sequence of the first region of the first template strand, wherein each of said reversibly-terminated nucleotides comprises a fluorescent label and a 3′ blocking group, wherein the blocking group prevents any further nucleotide incorporation into the 3′ end of the cleaved polynucleotide template to result in a synthesized polynucleotide, and wherein said sequential incorporation comprises:
 (i) incorporating one of said reversibly-terminated nucleotides into the first free 3′ hydroxyl group and detecting a fluorescent signal generated from the fluorescent label using a CCD camera or other fluorescence detection means, and 
 (ii) cleaving the fluorescent label and the 3′ blocking group from said one of said reversibly-terminated nucleotides in the 3′ end of said synthesized polynucleotide, thereby yielding a free 3′ hydroxyl group before another of said reversibly-terminated nucleotides is incorporated into the 3′ end of the synthesized polynucleotide, 
   (d) adding an unlabeled nucleotide to the free 3′ hydroxyl group in the last nucleotide added in step (c) and performing an extension reaction in the presence of different unlabeled nucleotides, thereby generating a synthesized polynucleotide template having a free 3′ hydroxyl group, wherein the second template strand is complementary to said first template strand and comprises a second region,   (e) cleaving the cleaved polynucleotide template, thereby yielding a second cleaved polynucleotide template comprising a 3′ end that comprises a second free 3′ hydroxyl group used for initiating sequencing of the second region of the synthesized polynucleotide template, wherein the cleaving comprises contacting the cleaved polynucleotide template with a nicking endonuclease to generate a nick at a site before, at, or beyond the 3′ end of the second hairpin linker;   (f) performing a second sequencing-by-synthesis reaction comprising sequential incorporation of different complementary reversibly-terminated nucleotides into the 3′ end generated in step (e), thereby determining the sequence of the second region of the synthesized polynucleotide template, wherein each of said reversibly-terminated nucleotides comprises a fluorescent label and a 3′ blocking group, wherein the blocking group prevents any further nucleotide incorporation into the 3′ end of the polynucleotide template to result in a second synthesized polynucleotide, and wherein said sequential incorporation comprises
 (i) incorporating one of said reversibly-terminated nucleotides into the second free 3′ hydroxyl group and detecting a fluorescent signal generated from the fluorescent label using a CCD camera or other fluorescence detection means, and 
 (ii) cleaving the fluorescent label and the 3′ blocking group from said one of said reversibly-terminated nucleotides in the 3′ end of said second synthesized polynucleotide, thereby yielding a free 3′ hydroxyl group before another of said reversibly-terminated nucleotides is incorporated into the 3′ end of said polynucleotide template; 
 wherein said first template strand ranges in length from 100 nucleotides to 1 kb. 
   
     
     
         3 . The method of  claim 2 , wherein the cleaving the polynucleotide template in step (b) comprises contacting the polynucleotide template with a nicking enzyme. 
     
     
         4 . The method of  claim 3 , wherein the first hairpin linker comprises a recognition site for the nicking enzyme, which directs cleavage by the nicking enzyme at a site before, at or beyond the 3′ end of the first hairpin linker. 
     
     
         5 . The method of  claim 2 , wherein the first sequencing-by-synthesis reaction of step (c) is carried out using a strand-displacing polymerase enzyme. 
     
     
         6 . The method of  claim 2 , wherein the cleaving the cleaved polynucleotide template in step (e) comprises contacting the circular polynucleotide template with a nicking enzyme. 
     
     
         7 . The method of  claim 6 , wherein the second hairpin linker comprises a recognition site for a nicking enzyme, which directs cleavage by the nicking enzyme at a site before, at or beyond the 3′ end of the second hairpin linker. 
     
     
         8 . The method of  claim 2 , wherein the second sequencing-by-synthesis reaction of step (f) is carried out using a strand-displacing polymerase enzyme. 
     
     
         9 . The method of  claim 2 , wherein the polynucleotide template forms part of an array. 
     
     
         10 . The method of  claim 2 , wherein the first sequencing-by-synthesis reaction of step (c) comprises incorporation of 10 to 200 consecutive nucleotides. 
     
     
         11 . The method of  claim 2 , wherein the second sequencing-by-synthesis reaction of step (f) comprises incorporation of 10 to 200 consecutive nucleotides. 
     
     
         12 . The method of  claim 2 , wherein the extension reaction of step (d) comprises incorporation of at least 100 consecutive nucleotides. 
     
     
         13 . The method of  claim 2 , wherein the extension reaction of step (d) comprises incorporation of at least 100 consecutive nucleotides. 
     
     
         14 . The method of  claim 2 , wherein the extension reaction of step (d) is performed without determining the identity of the unlabeled nucleotide add in step (d). 
     
     
         15 . The method of  claim 2 , wherein the polynucleotide template is directly linked to the surface of a solid support.