MODIFIED TRANSPOSASES FOR IMPROVED INSERTION SEQUENCE BIAS AND INCREASED DNA INPUT TOLERANCE
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Abstract
Presented herein are transposase enzymes and reaction conditions for improved fragmentation and tagging of nucleic acid samples, in particular altered transposases and reaction conditions which exhibit improved insertion sequence bias, as well as methods and kits using the same.
86 Citations
21 Claims
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1. (canceled)
- 2. A mutant Tn5 transposase modified relative to a wild type Tn5 transposase, the mutant transposase comprising one or more mutations at position Lys212 and/or Pro214 and/or Ala338.
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17. A method for sequencing a target DNA, comprising:
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(a) incubating the target DNA with transposome complexes comprising (1) a mutant Tn5 transposase, wherein the mutant Tn5 transposase is modified relative to a wild type Tn5 transposase, the mutant transposase comprising one or more mutations at position Lys212 and/or Pro214 and/or Ala338; 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 domain, 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) optionally amplifying the fragments by providing a polymerase and an amplification primer corresponding to a portion of the first polynucleotide, thereby generating a representative library of di-tagged fragments having the first tag at the 5′
ends and a second tag at the 3′
ends;(d) providing first sequencing primers comprising a portion corresponding to the first sequencing tag domain; and (e) extending the first sequencing primers and detecting the identity of nucleotides adjacent to the first sequencing tag domains of the representative library of di-tagged fragments in parallel.
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18. A kit for performing an in vitro transposition reaction comprising:
- transposome complexes comprising
(1) a mutant Tn5 transposase, wherein the mutant Tn5 transposase is modified relative to a wild type Tn5 transposase, the mutant transposase comprising one or more mutations at position Lys212 and/or Pro214 and/or Ala338; and (2) a polynucleotide comprising a 3′
portion comprising a transposon end sequence.
- transposome complexes comprising
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21. A method of generating uniform fragment sizes across a range of target DNA input amounts, comprising:
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(a) providing a target DNA;
wherein the amount of target DNA is selected from a range of target DNA amounts, and(b) incubating the target DNA with transposome complexes comprising a mutant Tn5 transposase, wherein the mutant Tn5 transposase is modified relative to a wild type Tn5 transposase, the mutant transposase comprising one or more mutations at position Lys212 and/or Pro214 and/or Ala338; and
wherein said transposases fragments the target DNA to generate uniform fragment sizes.
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Specification