Method for sequencing a polynucleotide template
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. A method for pairwise sequencing of first and second regions of a target double-stranded polynucleotide, wherein said first and second regions are in complementary strands of the target polynucleotide, the method comprising:
(a) providing a solid support having immobilised thereon a plurality of template polynucleotide duplexes each comprising the 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;
(b) cleaving the second template strands of a sub-fraction of the template polynucleotide duplexes to remove all or a portion of said second template strands, thereby generating single-stranded regions on the complementary first template strands;
(c) hybridising first sequencing primers to the single-stranded regions of the first template strands generated in part (b);
(d) carrying out a first sequencing reaction by sequential addition of nucleotides to the first sequencing primer to determine the sequence of a first region of the target polynucleotide in the first template strand;
(e) cleaving the first template strands of substantially all intact template polynucleotide duplexes not cleaved in part (b) to remove all or a portion of said first template strands, thereby generating single-stranded regions on the second template strands that were not cleaved in part (b);
(f) hybridising a second sequencing primer to the single stranded regions of the second template strands generated in part (e); and
(g) carrying out a second sequencing reaction by sequential addition of nucleotides to second sequencing primer to determine the sequence of a second region of the target polynucleotide in the second template strand.
2. The method according to claim 1 wherein the sub-fraction of the template polynucleotide duplexes cleaved in step (b) is approximately half.
3. The method according to claim 1 wherein the plurality of template polynucleotide duplexes provided in step (a) comprises a mixture of first and second template polynucleotide duplexes each comprising the same double-stranded target polynucleotide, wherein a first template strand of both the first and second template polynucleotide duplexes includes a cleavage site Y and 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 to cleavage site Y;
step (b) comprises cleaving the second template strand of the first template polynucleotide duplexes at cleavage site X;
and step (e) comprises cleaving the first template strand of both the first and second template polynucleotide duplexes at cleavage site Y.
4. The method according to claim 3 , wherein cleavage site X permits enzymatic, chemical or photochemical cleavage of the second template strand of the first template polynucleotide duplexes.
5. The method according to claim 3 , wherein cleavage site Y permits enzymatic, chemical or photochemical cleavage of the first template strand of both the first and second template polynucleotide duplexes.
6. The method according to claim 5 , wherein cleavage site Y comprises a diol linkage which is cleaved by treatment with a chemical cleavage agent comprising periodate.
7. The method according to claim 3 , wherein cleavage site X is a site for enzymatic cleavage and cleavage site Y is a site for chemical or photochemical cleavage.
8. The method according to claim 7 , wherein cleavage site X is a site for cleavage with a restriction endonuclease.
9. The method according to claim 3 , wherein the mixture of first and second template polynucleotide duplexes is formed by an amplification reaction using a combination of amplification primers immobilised 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.
10. The method according to claim 1 , wherein the plurality of template polynucleotide duplexes of step (a) are present on a clustered array.
11. The method according to claim 10 , wherein the clustered array is formed by solid-phase nucleic acid amplification.
12. The method according to claim 10 , wherein the plurality of template polynucleotide duplexes of step (a) are present within a single cluster on said clustered array.
13. The method according to claim 12 , wherein each individual cluster on said clustered array comprises first and second polynucleotide duplexes derived from amplification of a single target polynucleotide molecule.
14. The method according to claim 12 , wherein at least 95% of the clusters on the array comprise template polynucleotide duplexes derived from amplification of different single target polynucleotides.