Transposon end compositions and methods for modifying nucleic acids

摘要

Compositions of transposome complexes for generating DNA fragments with specific 5′- and 3′-tags. Kits for generating libraries for sequencing, with transposome complexes, enzymes, oligonucleotides or other components.

主权项

1. A method for sequencing a target DNA, comprising:
(a) incubating the target DNA with transposome complexes comprising
(1) a transposase and(2) a first polynucleotide comprising
(i) a 3′ portion comprising a transposon end sequence, and(ii) a first tag comprising a first sequencing tag,under conditions whereby the target DNA is fragmented, and the 3′ transposon end sequence of the first polynucleotide is transferred to the 5′ ends of the fragments, thereby producing double-stranded fragments wherein the 5′ ends are tagged with the first tag, and there is a single-stranded gap at the 3′ ends of the 5′-tagged strands; (b) incubating the fragments with a nucleic acid modifying enzyme under conditions whereby a second tag is attached to the 3′ ends of the 5′-tagged strands; (c) amplifying the fragments by providing a polymerase and an amplification primer comprising a capture tag and a sequence complementary to a portion of the second tag, thereby generating amplified fragments having capture tags, the first tag at the 5′ ends and the second tag at the 3′ ends, wherein the amplification is non-selective such that a population of amplified fragments representative of the target DNA is generated; (d) capturing the amplified fragments to a surface through the capture tags; and (e) simultaneously sequencing the captured amplified fragments on the surface in a single multiplex format by providing first sequencing primers comprising a portion corresponding to the first sequencing tag, extending the first sequencing primers, and detecting the identity of nucleotides adjacent to the first sequencing tags.