Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

• US 6,376,181 B2
• Filed: 12/14/1999
• Issued: 04/23/2002
• Est. Priority Date: 04/28/1997
• Status: Expired due to Fees

• Current Assignee: UT-Battelle LLC
• Original Assignee: UT-Battelle LLC
• Inventors: Foote, Robert S., Ramsey, J. Michael
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/460,316
• Publication Number:
  US 20010006785A1
• Time in Patent Office: 861 Days
• Field of Search: 435/6, 436/94
• US Class Current: 435/6.19
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00831 Glass
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• 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 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

Claims

1. A method for analyzing nucleic acids comprising the steps of:

a) providing a substrate having a microchannel structure which includes at least one microchannel therein;

b) immobilizing a number of different nucleic acid probes within at least a portion of said microchannel structure, at least one said microchannel having a probe-containing portion with a like number of probe sites, wherein each of said different nucleic acid probes is immobilized at a discrete probe site;

c) moving a target nucleic acid sample under the influence of an electrokinetic force into the probe-containing portion of said microchannel;

d) subjecting said target nucleic acid sample in said probe-containing portion of said microchannel to hybridization conditions;

e) labeling with a fluorescent substance one member selected from the group consisting of said target nucleic acid sample and any hybrids formed in step d; and

f) detecting fluorescence emission from said fluorescent substance.

2. A method according to claim 1, wherein the step of moving said target nucleic acid sample includes electroosmotically transporting a solution containing said target nucleic acid sample through said microchannel structure.

3. A method according to claim 1, wherein the step of moving said target nucleic acid sample includes electrophoretically migrating said target nucleic acid sample through a solution contained in said microchannel structure.

4. A method according to claim 1, wherein said microchannel structure is in fluid communication with at least two fluid reservoirs and the step of moving said target nucleic acid includes applying electrical voltages to said at least two fluid reservoirs, one of said reservoirs containing a solution of said target nucleic acid sample.

5. A method according to claim 1, wherein any hybrids formed in step d) are labeled with a fluorescent double-strand-specific nucleic acid stain in step e).

6. A method according to claim 1, wherein said fluorescent substance is electrokinetically transported to said probe-containing portion of said microchannel.

7. A method according to claim 1, additionally comprising the step of washing said probe-containing portion of said microchannel before step e) to remove unhybridized target nucleic acid sample.

8. A method according to claim 6, additionally comprising the step of washing said probe-containing portion of said microchannel after step e) to remove fluorescent substance that is not incorporated into hybrids formed in step d).

9. A method according to claim 8, wherein said washing step comprises electrokinetically transporting buffer through said probe-containing portion of said microchannel probe.