High efficiency multiplexed nucleic acid capture in a structured microenvironment
First Claim
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1. A method for sample analysis, comprising:
- a) enzymatically attaching a reactive group to nucleic acid molecules in a sample;
b) covalently reacting said reactive group with reactive sites in a porous support, thereby covalently tethering said nucleic acid molecules to said porous support, wherein the porous support is a silica membrane;
c) performing a primer extension reaction using said tethered nucleic acid molecules as a template to produce primer extension products, wherein the primer extension is done using one or more gene-specific primers that hybridize to a subset of the nucleic acid molecules that are tethered to the porous support but do not hybridize to an adaptor sequence; and
d) eluting the primer extension products from the porous support, while leaving the tethered nucleic acid molecules tethered to said porous support, wherein said eluting is done by;
(i) treating the product of step c) with heat or a chaotropic agent to denature the primer extension products from the tethered nucleic acid molecules; and
(ii) applying a force that separates the primer extension products and the porous support.
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Abstract
Provided herein is a method for sample analysis. In some embodiments, the method may involve: a) enzymatically attaching a reactive group to nucleic acid molecules in a sample; b) covalently reacting the reactive group with surface exposed reactive sites on a porous support, thereby covalently tethering the nucleic acid molecules to the porous support; c) performing a primer extension reaction using the tethered nucleic acid molecules as a template to produce primer extension products; and d) eluting the primer extension products from the porous support, while leaving the tethered nucleic acid molecules tethered to the porous support.
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14 Claims
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1. A method for sample analysis, comprising:
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a) enzymatically attaching a reactive group to nucleic acid molecules in a sample; b) covalently reacting said reactive group with reactive sites in a porous support, thereby covalently tethering said nucleic acid molecules to said porous support, wherein the porous support is a silica membrane; c) performing a primer extension reaction using said tethered nucleic acid molecules as a template to produce primer extension products, wherein the primer extension is done using one or more gene-specific primers that hybridize to a subset of the nucleic acid molecules that are tethered to the porous support but do not hybridize to an adaptor sequence; and d) eluting the primer extension products from the porous support, while leaving the tethered nucleic acid molecules tethered to said porous support, wherein said eluting is done by; (i) treating the product of step c) with heat or a chaotropic agent to denature the primer extension products from the tethered nucleic acid molecules; and (ii) applying a force that separates the primer extension products and the porous support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification