Methods for sequencing a polynucleotide template
First Claim
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1. A method for sequencing a first region and a second region of a polynucleotide template, the method comprising:
- (a) providing a polynucleotide template immobilized on a surface, said polynucleotide template comprisinga single polynucleotide strand having a first region and a second region, said first region and said second region separated by at least 50 nucleotides, anda self-complementary hairpin polynucleotide linker comprising a loop region and a stem region, wherein the 5′
end of one strand of the stem region is linked to the 3′
end of said polynucleotide strand and the 3′
end of the other strand of the stem region comprises a first free 3′
-hydroxyl group used for initiating sequencing of the first region of the polynucleotide strand,(b) performing a first sequencing-by-synthesis reaction comprising sequential incorporation of different complementary reversibly-terminated nucleotides into the 3′
end of said polynucleotide template, thereby determining the sequence of the first region of the polynucleotide strand, wherein each of said nucleotides comprises a fluorescent label and a 3′
blocking group, the blocking group prevents any further nucleotide incorporation into the 3′
end of said polynucleotide template, and wherein said sequential incorporation comprises(i) incorporating one of said 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 nucleotides in the 3′
end of said polynucleotide template, thereby yielding a free 3′
-hydroxyl group before another of said nucleotides is incorporated into the 3′
end of the polynucleotide template,(c) adding an unlabeled nucleotide to the free 3′
-hydroxyl group in the last nucleotide added in step (b) and performing an extension reaction in the presence of different unlabeled nucleotides, thereby generating an extension product having a second free 3′
hydroxyl group used for initiating sequencing of the second region of the polynucleotide strand, and(d) performing a second sequencing-by-synthesis reaction comprising sequential incorporation of different complementary reversibly-terminated nucleotides into the 3′
end of the extension product generated in step (c), thereby determining the sequence of the second region of the polynucleotide strand, wherein each of said nucleotides comprises a fluorescent label and a 3′
blocking group, the blocking group prevents any further nucleotide incorporation into the 3′
end of said polynucleotide template, and wherein said sequential incorporation after step (c) comprisesi) incorporating one of said 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 nucleotides in the 3′
end of said extension product, thereby yielding a free 3′
hydroxyl group before another of said nucleotides is incorporated into the 3′
end of said extension product;
wherein said polynucleotide strand ranges in length from 100 nucleotides to 1 kb.
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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.
62 Citations
9 Claims
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1. A method for sequencing a first region and a second region of a polynucleotide template, the method comprising:
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(a) providing a polynucleotide template immobilized on a surface, said polynucleotide template comprising a single polynucleotide strand having a first region and a second region, said first region and said second region separated by at least 50 nucleotides, and a self-complementary hairpin polynucleotide linker comprising a loop region and a stem region, wherein the 5′
end of one strand of the stem region is linked to the 3′
end of said polynucleotide strand and the 3′
end of the other strand of the stem region comprises a first free 3′
-hydroxyl group used for initiating sequencing of the first region of the polynucleotide strand,(b) performing a first sequencing-by-synthesis reaction comprising sequential incorporation of different complementary reversibly-terminated nucleotides into the 3′
end of said polynucleotide template, thereby determining the sequence of the first region of the polynucleotide strand, wherein each of said nucleotides comprises a fluorescent label and a 3′
blocking group, the blocking group prevents any further nucleotide incorporation into the 3′
end of said polynucleotide template, and wherein said sequential incorporation comprises(i) incorporating one of said 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 nucleotides in the 3′
end of said polynucleotide template, thereby yielding a free 3′
-hydroxyl group before another of said nucleotides is incorporated into the 3′
end of the polynucleotide template,(c) adding an unlabeled nucleotide to the free 3′
-hydroxyl group in the last nucleotide added in step (b) and performing an extension reaction in the presence of different unlabeled nucleotides, thereby generating an extension product having a second free 3′
hydroxyl group used for initiating sequencing of the second region of the polynucleotide strand, and(d) performing a second sequencing-by-synthesis reaction comprising sequential incorporation of different complementary reversibly-terminated nucleotides into the 3′
end of the extension product generated in step (c), thereby determining the sequence of the second region of the polynucleotide strand, wherein each of said nucleotides comprises a fluorescent label and a 3′
blocking group, the blocking group prevents any further nucleotide incorporation into the 3′
end of said polynucleotide template, and wherein said sequential incorporation after step (c) comprisesi) incorporating one of said 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 nucleotides in the 3′
end of said extension product, thereby yielding a free 3′
hydroxyl group before another of said nucleotides is incorporated into the 3′
end of said extension product;wherein said polynucleotide strand ranges in length from 100 nucleotides to 1 kb. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Current AssigneeIllumina Cambridge Limited (Illumina Incorporated)
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Original AssigneeIllumina Cambridge Limited (Illumina Incorporated)
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InventorsSwerdlow, Harold Philip
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Primary Examiner(s)Lu, Frank
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Application NumberUS11/989,170Publication NumberTime in Patent Office4,079 DaysField of Search435 61, 435 611, 435 612, 435 911, 435 912, 4352831, 4352871, 4352872, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253US Class CurrentCPC Class CodesC12Q 1/6853 using modified primers or t...C12Q 1/6869 Methods for sequencingC12Q 2525/119 incorporating abasic sitesC12Q 2525/301 Hairpin oligonucleotidesC12Q 2533/101 Primer extensionC12Q 2535/119 Double strand sequencingC12Q 2565/525 characterised by the captur...C12Q 2565/537 characterised by the captur...
