Method for sequencing a polynucleotide template

Abstract

The invention relates to methods for pairwise sequencing of a double-stranded polynucleotide template, which permit the sequential determination of nucleotide sequences in two distinct and separate regions on complementary strands of the double-stranded polynucleotide template. The two regions for sequence determination may or may not be complementary to each other.

1 . (canceled) 
     
     
         2 . A complex for pairwise sequencing of first and second regions of a target double-stranded polynucleotide, the complex comprising:
 (a) a solid support;   (b) a plurality of template polynucleotide duplexes each comprising a double-stranded target polynucleotide, wherein each template duplex is formed from complementary first and second template strands linked to the solid support at their 5′ ends, wherein the plurality of template polynucleotide duplexes are in a single colony on the solid support; and   (c) a plurality of single-stranded first template strands in the colony linked to the solid support at their 5′ ends.   
     
     
         3 . The complex of  claim 2 , wherein the plurality of template polynucleotide duplexes comprises a mixture of first and second template polynucleotide duplexes, each comprising the same double-stranded target polynucleotide,
 wherein the first template strand of both the first and second template polynucleotide duplexes includes a cleavage site Y, and   wherein a second template strand of the first template polynucleotide duplexes but not the second template polynucleotide duplexes includes a cleavage site X, which is different from the cleavage site Y.   
     
     
         4 . The complex of  claim 2 , wherein the plurality of template polynucleotide duplexes are present on a clustered array. 
     
     
         5 . The complex of  claim 2 , wherein the first and second template polynucleotide duplexes are formed by an amplification reaction using a combination of amplification primers immobilized on said solid support, wherein said combination includes first and second forward primers and reverse primers having the structures:
 First forward primer: A-L-X-S1,   Second forward primer: A-L-S1,   Reverse primer: A-L-Y-S2,   wherein A is a moiety which allows attachment to the solid support, L is an optional linker sequence, X is a cleavage site, Y is a cleavage site different to X, S1 and S2 are sequences which permit amplification of a nucleic acid molecule comprising the target double-stranded polynucleotide.