Sample preparation on a solid support
First Claim
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1. A method of preparing an immobilized library of tagged DNA fragments comprising:
- (a) providing a solid support having transposome complexes immobilized thereon, wherein said transposome complexes comprise a transposase bound to a first polynucleotide, said first polynucleotide comprising(i) a 3′
portion comprising a transposon end sequence, and(ii) a first tag comprising a first tag domain;
(b) providing a sample comprising target DNA, proteins and other cellular components from an in vivo source;
(c) applying said sample to the solid support under conditions wherein said target DNA, proteins and other cellular components are present at the same proportion as in said in vivo source, whereby the target DNA is fragmented by the transposome complexes, and the 3′
transposon end sequence of the first polynucleotide is transferred to a 5′
end of at least one strand of the fragments;
thereby producing an immobilized library of double-stranded fragments wherein at least one strand is 5′
-tagged with the first tag.
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Abstract
Presented are methods and compositions for using immobilized transposase and a transposon end for generating an immobilized library of 5′-tagged double-stranded target DNA on a surface. The methods are useful for generating 5′- and 3′-tagged DNA fragments for use in a variety of processes, including massively parallel DNA sequencing.
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Citations
27 Claims
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1. A method of preparing an immobilized library of tagged DNA fragments comprising:
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(a) providing a solid support having transposome complexes immobilized thereon, wherein said transposome complexes comprise a transposase bound to a first polynucleotide, said first polynucleotide comprising (i) a 3′
portion comprising a transposon end sequence, and(ii) a first tag comprising a first tag domain; (b) providing a sample comprising target DNA, proteins and other cellular components from an in vivo source; (c) applying said sample to the solid support under conditions wherein said target DNA, proteins and other cellular components are present at the same proportion as in said in vivo source, whereby the target DNA is fragmented by the transposome complexes, and the 3′
transposon end sequence of the first polynucleotide is transferred to a 5′
end of at least one strand of the fragments;
thereby producing an immobilized library of double-stranded fragments wherein at least one strand is 5′
-tagged with the first tag. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification