Uniform fragmentation of DNA using binding proteins
First Claim
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1. A method for sequencing a plurality of polynucleotides comprising:
- (a) binding a plurality of protection molecules to a plurality of polynucleotides;
(b) cleaving polynucleotides in the plurality of polynucleotides that are bound to the protection molecules, thereby generating a plurality of polynucleotide fragments of substantially uniform size;
(c) amplifying polynucleotide fragments of the plurality of polynucleotide fragments on a solid support, thereby producing an array of locations, each location comprising multiple copies of a single polynucleotide fragment of the plurality of polynucleotide fragments; and
(d) performing paired-end sequencing on the copies of the polynucleotide fragments at a plurality of the locations of the array, wherein the paired end sequencing comprises sequencing a first strand of each of the copies, converting the first strand into a double-stranded template and sequencing a second strand of the double-stranded template.
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Abstract
The invention provides a method for preparing and analysing a population of fragmented polynucleotide sequences having a substantially uniform size. The method can include steps of (a) binding at least one protection molecule to at least one polynucleotide sequence; (b) cleaving the at least one polynucleotide sequence to generate a plurality of polynucleotide fragment sequences of substantially uniform size; (c) amplifying the polynucleotide fragments; and (d) determining a sequence characteristic of a plurality of the polynucleotide fragments.
20 Citations
17 Claims
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1. A method for sequencing a plurality of polynucleotides comprising:
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(a) binding a plurality of protection molecules to a plurality of polynucleotides; (b) cleaving polynucleotides in the plurality of polynucleotides that are bound to the protection molecules, thereby generating a plurality of polynucleotide fragments of substantially uniform size; (c) amplifying polynucleotide fragments of the plurality of polynucleotide fragments on a solid support, thereby producing an array of locations, each location comprising multiple copies of a single polynucleotide fragment of the plurality of polynucleotide fragments; and (d) performing paired-end sequencing on the copies of the polynucleotide fragments at a plurality of the locations of the array, wherein the paired end sequencing comprises sequencing a first strand of each of the copies, converting the first strand into a double-stranded template and sequencing a second strand of the double-stranded template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for sequencing a plurality of polynucleotides comprising:
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(a) binding a plurality of protection molecules to a plurality of polynucleotides; (b) cleaving polynucleotides in the plurality of polynucleotides that are bound to the protection molecules, thereby generating a plurality of polynucleotide fragments of substantially uniform size; (c) amplifying polynucleotide fragments of the plurality of polynucleotide fragments on a solid support, thereby producing an array of locations, each location comprising multiple copies of a single polynucleotide fragment of the plurality of polynucleotide fragments; and (d) performing paired-end sequencing on the copies of the polynucleotide fragments at a plurality of the locations of the array, wherein the paired end sequencing comprises a step of strand resynthesis between a first sequencing read of the copies and a second read of the copies wherein the strand resynthesis is performed using multiple cycles of hybridization, extension and denaturation.
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Specification