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US 10,240,196 B2Granted
Transposase-random priming DNA sample preparation
AssigneeAgilent Technologies, Inc.
InventorsBahram Arezi, Michael Borns, Holly Hogrefe, Connie Hansen
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Abstract
Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.
Claims17
A method comprising:
- (a) contacting a sample comprising double stranded DNA fragments having an average length of from 150 bp to 1.5 Kb with a plurality of transposase duplexes each loaded with an adapter to produce adapter-tagged fragments, wherein the adapter comprises a duplex region comprising a transposase recognition sequence and a 5′ overhang region comprising a first primer sequence;
- (b) performing a primer extension reaction on the adapter tagged fragments using a random primer to produce random primer extension products, wherein the random primer comprises a random 3′ sequence and a 5′ sequence comprising a second primer sequence;
- (c) amplifying the random primer extension products of (b) by polymerase chain reaction (PCR) using a forward primer comprising the first primer sequence at its 3′ end and a reverse primer comprising the second primer sequence at its 3′ end to produce PCR amplification products.
A method comprising:
- (a) contacting a sample comprising double stranded DNA fragments having an average length of less than 1 kb with a plurality of transposase duplexes each loaded with an adapter to produce adapter-tagged fragments, wherein the adapter comprises a duplex region comprising a transposase recognition sequence and a 5′ overhang region comprising a first primer sequence;
- (b) adding an oligo-dN tail to the top strand of the adapter-tagged fragments using an enzyme with terminal transferase activity to produce tailed adapter-tagged fragments;
- (c) performing a primer extension reaction on the tailed adapter-tagged fragments using a tail primer to produce tail primer extension products, wherein the tail primer comprises a 3′ sequence that hybridizes to the oligo-dN tail and a 5′ sequence comprising a second primer sequence;
- (d) amplifying the tail primer extension products of (c) by polymerase chain reaction (PCR) using a forward primer comprising the first primer sequence at its 3′ end and a reverse primer comprising the second primer sequence at its 3′ end to produce PCR amplification products.
1 more element
Dependencies are inferred from the claim text.