Microfluidic method for nucleic acid purification and processing

• US 6,344,326 B1
• Filed: 02/10/2000
• Issued: 02/05/2002
• Est. Priority Date: 07/30/1996
• Status: Expired due to Term

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Aclara Biosciences, Inc (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Nelson, Robert J., Williams, Stephen J., Hooper, Herbert H., Hauser, Alan K., Sassi, Alexander P.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/501,706
• Time in Patent Office: 726 Days
• Field of Search: 435/6, 435/91.2, 435/7.93, 536/25.32, 536/24.4, 536/25.4
• US Class Current: 435/6.12
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/5002 Partitioning blood components
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses

Abstract

Integrated microfluidic devices comprising at least an enrichment channel (10) and a main electrophoretic flowpath (12) 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 (6). 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.

Claims

1. A method for nucleic acid sample clean-up using an enrichment channel and a discharge outlet formed in a substrate, said enrichment channel containing an enrichment medium and an affinity binding pair having first and second binding members, said first binding member having an oligonucleotide moiety that is complementary in base sequence to a nucleic acid portion of said sample mixture, said second binding member being carried on a solid support in said medium, said method comprising the steps of:

introducing a nucleic acid mixture having a nucleic acid portion and a waste portion into the enrichment channel,

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 bound entities between said nucleic acid portion and said moiety,

contacting in the enrichment medium bound entities with the second binding member of the affinity binding pair, 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

releasing the nucleic acid portion of said captured bound entities with a competitive displacing member whose binding affinity for the second binding member is greater than that of the first binding member to yield a purified nucleic acid portion.

2. The method according to claim 1 wherein the combining step is performed after the introducing step.

3. The method according to claim 1 wherein the introducing step is performed after the combining step.

4. The method according to claim 1 further comprising the step of nucleic acid sequencing the purified nucleic acid portion.

5. The method according to claim 4 wherein the nucleic acid sequencing step includes the step of dideoxy enzymatic chain-termination sequencing the purified nucleic acid portion.

6. The method according to claim 1 wherein the first binding member includes modified biotin having a lower affinity than biotin to the second binding member.

7. The method according to claim 6 wherein the modified biotin is dethiobiotin.

8. The method according to claim 6 wherein the modified biotin is a dethiobiotin derivative.

9. The method according to claim 1 wherein the second binding member includes an avidin-based protein.

10. The method according to claim 1 wherein the solid support includes a plurality of magnetic bodies.

11. The method according to claim 1 wherein the solid support includes a plurality of paramagnetic bodies.

12. The method according to claim 1 wherein the competitive displacing member comprises biotin.

13. The method according to claim 1 wherein the nucleic acid portion of the nucleic acid sample mixture includes a DNA template and a sequencing primer.

14. The method of claim 8, wherein the nucleic acid portion of the nucleic acid sample mixture includes a terminal dideoxynucleotide.

15. The method according to claim 8 wherein the nucleic acid portion of the nucleic acid sample mixture includes a deoxynucleotide.

16. The method according to claim 8 wherein said first binding member is dethiobiotin and wherein said second binding member is an avidin based protein and said competitive displacing member is biotin, further comprising the step of performing PCR amplification on the purified nucleic acid portion.