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US 10,017,759 B2Granted
Library preparation of tagged nucleic acid
AssigneeIllumina, Inc.
InventorsFiona Kaper, Gordon Cann
Life of this patent
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Abstract
A method of preparing a library of tagged nucleic acid fragments including contacting a population of cells directly with a lysis reagent having one or more protease to generate a cell lysate; inactivating the protease to generate an inactivated cell lysate, and applying a transposase and a transposon end composition containing a transferred strand to the inactivated cell lysate under conditions wherein the target nucleic acid and the transposon end composition undergo a transposition reaction.
Claims23
A method of preparing a library of tagged nucleic acid fragments comprising:
- (a) contacting a population of cells consisting of a single cell, directly with a lysis reagent to generate a cell lysate, wherein the lysis reagent comprises one or more proteases, and wherein the cell lysate contains a target nucleic acid;
- (b) inactivating the one or more proteases to form an inactivated cell lysate;
- and
- (c) directly applying at least one transposase and at least one transposon end composition containing a transferred strand to the inactivated cell lysate under conditions where the target nucleic acid and the transposon end composition undergo a transposition reaction to generate a mixture;
- wherein the target nucleic acid comprises double-stranded DNA;
- wherein:
- (i) the target nucleic acid is fragmented to generate a plurality of target nucleic acid fragments, and
- (ii) the transferred strand of the transposon end composition is joined to 5′ ends of each of a plurality of the target nucleic acid fragments to generate a plurality of 5′ tagged target nucleic acid fragments;
- wherein the target nucleic acid remains double-stranded DNA for the duration of (a) through (c);
- and
- wherein no DNA purification or amplification occurs between (a) and (c).
8 more elements
Dependencies are inferred from the claim text.