Microfluidic method for nucleic acid purification and processing
First Claim
1. A method for nucleic acid sample clean-up using a substrate having a surface and an enrichment microchannel, a main electrophoretic flowpath and a discharge outlet, formed in the substrate, said enrichment microchannel comprising an enrichment region and said main electrophoretic flowpath comprising a working region, said enrichment microchannel being in fluid transfer relationship with said main electrophoretic flowpath, and an affinity binding pair having complementary first and second binding members, said first binding member comprising a label, said method comprising the steps of:
- introducing a nucleic acid mixture having a nucleic acid portion and a waste portion into the enrichment microchannel,combining the first binding member of the affinity binding pair with the nucleic acid portion of at least some of the nucleic acid mixture to form a labeled nucleic acid portion of bound entities between nucleic acid molecules of said nucleic acid portion and said labeled first binding member,contacting in the enrichment region bound entities with the second binding member of the affinity binding pair, which is bound to at least one solid support to capture at least a part of the bound entities to form a captured nucleic acid portion,washing the captured nucleic acid portion to direct the waste portion and the nucleic acid portion excluding the captured nucleic acid portion through said discharge outlet and away from the working region,releasing the captured nucleic acid portion by competitive displacement of the first binding member bound to the captured nucleic acid portion with a competitive displacing member having a higher affinity for the second binding member than the first binding member to yield a purified nucleic acid portion, andtransporting the purified nucleic acid portion to the working region,whereby the purified nucleic acid portion is processed or analyzed or processed and analyzed in the working region.
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Abstract
Integrated microfluidic devices comprising at least an enrichment channel and a main electrophoretic flowpath are provided. In the subject integrated devices, the enrichment channel and the main electrophoretic flowpath are positioned so that waste fluid flows away from said main electrophoretic flowpath through a discharge outlet. The subject devices find use in a variety of electrophoretic applications, including clinical assays, high throughput screening for genomics and pharmaceutical applications, point-or-care in vitro diagnostics, molecular genetic analysis and nucleic acid diagnostics, cell separations, and bioresearch generally.
523 Citations
18 Claims
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1. A method for nucleic acid sample clean-up using a substrate having a surface and an enrichment microchannel, a main electrophoretic flowpath and a discharge outlet, formed in the substrate, said enrichment microchannel comprising an enrichment region and said main electrophoretic flowpath comprising a working region, said enrichment microchannel being in fluid transfer relationship with said main electrophoretic flowpath, and an affinity binding pair having complementary first and second binding members, said first binding member comprising a label, said method comprising the steps of:
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introducing a nucleic acid mixture having a nucleic acid portion and a waste portion into the enrichment microchannel, combining the first binding member of the affinity binding pair with the nucleic acid portion of at least some of the nucleic acid mixture to form a labeled nucleic acid portion of bound entities between nucleic acid molecules of said nucleic acid portion and said labeled first binding member, contacting in the enrichment region bound entities with the second binding member of the affinity binding pair, which is bound to at least one solid support to capture at least a part of the bound entities to form a captured nucleic acid portion, washing the captured nucleic acid portion to direct the waste portion and the nucleic acid portion excluding the captured nucleic acid portion through said discharge outlet and away from the working region, releasing the captured nucleic acid portion by competitive displacement of the first binding member bound to the captured nucleic acid portion with a competitive displacing member having a higher affinity for the second binding member than the first binding member to yield a purified nucleic acid portion, and transporting the purified nucleic acid portion to the working region, whereby the purified nucleic acid portion is processed or analyzed or processed and analyzed in the working region. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification