Methods for hybridization analysis utilizing electrically controlled hybridization

• US 5,849,486 A
• Filed: 09/27/1995
• Issued: 12/15/1998
• Est. Priority Date: 11/01/1993
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Juncosa, Robert David, Jackson, Thomas Ratcliffe, O'Connell, James Patrick, Sosnowski, Ronald George, Heller, Michael James
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/534,454
• Time in Patent Office: 1,175 Days
• Field of Search: 435/6, 435/810, 436/501, 536/23.1, 536/24.1, 536/24.3-24.33, 536/25.3, 935/77, 935/78, 935/88
• US Class Current: 435/6.11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• 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 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00614 Delimitation of the attachment areas
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01J 2219/00731 : Saccharides
• CPC Class Codes: B01J 2219/00828 : Silicon wafers or plates
• CPC Class Codes: B01J 2219/00853 : Employing electrode arrangements
• CPC Class Codes: B01J 2219/0095 : Control aspects
• CPC Class Codes: B01J 2219/0097 : Optical sensors
• CPC Class Codes: B01L 2200/025 : Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/143 : Quality control, feedback systems
• CPC Class Codes: B01L 2300/0636 : Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 : Electrodes
• CPC Class Codes: B01L 2300/0816 : Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0838 : Capillaries
• CPC Class Codes: B01L 2300/0877 : Flow chambers
• CPC Class Codes: B01L 2400/0421 : electrophoretic flow
• 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: 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 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 : Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/515 : characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 40/12 : Libraries containing saccharides or polysaccharides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: G11C 13/04 : using optical elements ; using other beam accessed elements, e.g. electron or ion beam
• CPC Class Codes: G11C 19/00 : Digital stores in which the information is moved stepwise, e.g. shift registers
• CPC Class Codes: H01L 2224/32225 : the item being non-metallic, e.g. insulating substrate with or without metallisation
• CPC Class Codes: H01L 2224/45144 : Gold (Au) as principal constituent
• CPC Class Codes: H01L 2224/48227 : connecting the wire to a bond pad of the item
• CPC Class Codes: H01L 2224/48472 : the other connecting portion not on the bonding area also being a wedge bond, i.e. wedge-to-wedge
• CPC Class Codes: H01L 2224/73265 : Layer and wire connectors
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/09701 : Low temperature co-fired ceramic [LTCC]
• CPC Class Codes: H01L 2924/3011 : Impedance

Abstract

A system for performing molecular biological diagnosis, analysis and multistep and multiplex reactions utilizes a selfaddressable, selfassembling microelectronic system for actively carrying out controlled reactions in microscopic formats. Preferably, a fluidic system flow a sample across an active area of the biochip, increasing diagnostic efficiency. Preferably, the fluidic system includes aflow cell having a window. Pulsed activation of the electrodes of the biochip are advantageously employed with the fluidic system, permitting more complete sampling of the materials within the biological sample. An improved detection system utilizes a preferably coaxially oriented excitation fiber, such as a fiber optic, disposed within a light guide, such as a liquid light guide. In this way, small geometry systems may be fluorescently imaged. A highly automated DNA diagnostic system results. Perturbation of the fluorescence signal during electronic denaturation is detailed and analyzed for analytical and diagnostic purposes. Such fluorescence perturbation information is combined with other information to provide improved analysis. DNA fingerprinting uses hybridizing DNA fragments of a given length and a capture sequence at a test site and then determining the level of reverse bias necessary to affect the hybridization, such as to dehybridize, and determine the length of the DNA.

Claims

1. A method for hybridization analysis between a sample and a probe, the analysis utilizing an electronic stringency control device, comprising the steps of:

providing the sample and probe with a fluorescent label under hybridization conditions on the electronic stringency control device, forming a fluorescently labelled hybridization product,

monitoring the fluorescence from the hybridization product,

subjecting the hybridization product to varying electrophoretic force, and

analyzing the fluorescent signal from the flourescently labeled hybridization product for variance as a function of said varying electrophoretic force.

2. The method for hybridization analysis of claim 1 wherein the fluorescence is analyzed for the fluorescent perturbation value.

3. The method for hybridization analysis of claim 2 wherein the fluorescence perturbation value is measured for the onset value.

4. The method for hybridization analysis of claim 2 wherein the fluorescence perturbation value is measured for its height.

5. The method for hybridization analysis of claim 2 wherein the fluorescence perturbation value is measured for the slope.

6. The method for hybridization analysis of claim 1 wherein the fluorescence is analyzed for the power level of the perturbation.

7. The method for hybridization analysis of claim 1 further including the steps of:

determining a second measure of hybridization between the sample and the probe, and

combining the information obtained by the first analysis including the step of subjecting the hybridization product to the varying electrophoretic force in the second measure to provide a indication of hybridization.

8. The method for hybridization analysis of claim 7 wherein the second measure of hybridization includes determination of the electronic melting point.

9. The method for hybridization analysis of claim 1 wherein the fluorescent label is placed in proximity to the initial denaturation site.

10. The method for hybridization analysis of claims 1 or 9 wherein the fluorescent label is intercalated adjacent a single base mismatch site.

11. The method for hybridization analysis of claim 10 wherein the fluorescent label is ethidium bromide.

12. The method for hybridization analysis of claim 10 wherein the fluorescent label is acridine.

13. The method for hybridization analysis of claims 1 or 7 wherein the electrophoretic force is in an amount less than is necessary to effect dehybridization of the sample and the probe.

14. The method for hybridization analysis of claim 1 wherein the hybridization product is subject to an oscillating electrophoretic force.

15. The method for hybridization analysis of claim 7 wherein the fluorescent label is placed in proximity to the initial denaturation site.

16. The method for hybridization analysis of claim 13 wherein the electrophoretic force is in an amount less than is necessary to effect complete denaturation of the sample and the probe.

17. The method for hybridization analysis of claim 14 where the fluorescent signal from a mismatched sample and probe exhibits a relatively greater response to the oscillating electrophoretic force relative to a matched hybridization product.

18. The method for hybridization analysis of claim 1 wherein the probe is longer than a 20-mer.

19. The method for hybridization analysis of claim 1 further including the step of averaging of the fluorescent signal.

20. A method for hybridization analysis between a sample and a probe comprising the steps of:

providing the sample and probe with a fluorescent label under hybridization conditions, forming a fluorescently labeled hybridization product,

monitoring the fluorescence from the hybridization product,

subjecting the hybridization product to varying electrophoretic force, and

analyzing the fluorescent signal for variance as a function of said varying electrophoretic force to determine the fluorescence perturbation value.

21. The method for hybridization analysis of claim 20 wherein the fluorescence perturbation value is measured for the onset value.

22. The method for hybridization analysis of claim 20 wherein the fluorescence perturbation value is measured for its height.

23. The method for hybridization analysis of claim 20 wherein the fluorescence perturbation value is measured for the slope.

24. The method for hybridization analysis of claim 20 wherein the fluorescence is analyzed for the power level of the perturbation.

25. The method for hybridization analysis of claim 20 further including the steps of:

determining a second measure of hybridization between the sample and the probe, and

combining the information obtained by the first analysis including the step of subjecting the hybridization product to the varying electrophoretic force in the second measure to provide a indication of hybridization.

26. The method for hybridization analysis of claim 25 wherein the second measure of hybridization includes determination of the electronic melting point.

27. The method for hybridization analysis of claim 20 wherein the fluorescent label is placed in proximity to the initial denaturation site.

28. The method for hybridization analysis of claim 20 wherein the fluorescent label is intercalated adjacent a single based mismatch site.

29. The method for hybridization analysis of claims 27 or 28 wherein the fluorescent label is ethidium bromide.

30. The method for hybridization analysis of claims 27 or 28 wherein the fluorescent label is acridine.

31. The method for hybridization analysis of claim 20 wherein the electrophoretic force is in an amount less than is necessary to effect dehybridization of the sample and the probe.

32. The method for hybridization analysis of claim 20 wherein the hybridization product is subject to an oscillating electrophoretic force.

33. The method for hybridization analysis of claim 25 wherein the fluorescent label is placed in proximity to the initial denaturation site.

34. The method for hybridization analysis of claim 31 wherein the electrophoretic force is in an amount less than is necessary to effect complete denaturation of the sample and the probe.

35. The method for hybridization analysis of claim 32 where the fluorescent signal from a mismatch sample and probe exhibits a relatively greater response to the oscillating electrophoretic force relative to a matched hybridization product.

36. The method for hybridization analysis of claim 20 wherein the probe is longer than a 20-mer.

37. The method for hybridization analysis of claim 20 further including the step of averaging of the fluorescent signal.