Method for sequencing a polynucleotide template
First Claim
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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.
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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.
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Citations
14 Claims
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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:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification