Methods and procedures for molecular biological analysis and diagnostics

• US 6,051,380 A
• Filed: 12/05/1997
• Issued: 04/18/2000
• Est. Priority Date: 11/01/1993
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Tu, Eugene, Heller, Michael J., Sosnowski, Ronald G., Butler, William F., Nerenberg, Michael I., Edman, Carl F.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/986,065
• Time in Patent Office: 865 Days
• Field of Search: 435/6, 435/810, 436/501, 536/25.3, 422/50, 422/68.1, 935/77, 935/78
• 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 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• 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 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• 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: B01L 2200/0647 : Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 : Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0636 : Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 : Electrodes
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 : electrophoretic flow
• CPC Class Codes: B01L 3/502707 : characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• 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 3/502761 : 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: 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 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6832 : Enhancement of hybridisation reaction
• 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 2224/95145 : Electrostatic alignment, i.e. polarity alignment with Coulomb charges
• CPC Class Codes: H01L 2224/95147 : by molecular lock-key, e.g. by DNA
• 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 29/6656 : using multiple spacer layers, e.g. multiple sidewall spacers
• CPC Class Codes: H01L 29/6659 : with both lightly doped source and drain extensions and source and drain self-aligned to the sides of the gate, e.g. lightly doped drain [LDD] MOSFET, double diffused drain [DDD] MOSFET
• CPC Class Codes: H01L 29/66628 : recessing the gate by forming single crystalline semiconductor material at the source or drain location
• CPC Class Codes: H01L 29/7834 : with a non-planar structure, e.g. the gate or the source or the drain being non-planar
• 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 2924/01019 : Potassium [K]
• CPC Class Codes: H01L 2924/01025 : Manganese [Mn]
• CPC Class Codes: H01L 2924/01046 : Palladium [Pd]
• CPC Class Codes: H01L 2924/01078 : Platinum [Pt]
• CPC Class Codes: H01L 2924/01079 : Gold [Au]
• CPC Class Codes: H01L 2924/10253 : Silicon [Si]

Abstract

A self-addressable, self-assembling microelectronic device is designed and fabricated to actively carry out and control multi-step and multiplex molecular biological reactions in microscopic formats. These reactions include nucleic acid hybridizations, antibody/antigen reactions, diagnostics, and biopolymer synthesis. The device can be fabricated using both microlithographic and micro-machining techniques. The device can electronically control the transport and attachment of specific binding entities to specific microlocations. The specific binding entities include molecular biological molecules such as nucleic acids and polypeptides. The device can subsequently control the transport and reaction of analytes or reactants at the addressed specific microlocations. The device is able to concentrate analytes and reactants, remove non-specifically bound molecules, provide stringency control for DNA hybridization reactions, and improve the detection of analytes. The device can be electronically replicated.

Claims

1. A method for transport and hybridization of DNA in an active electronic system comprising the steps of:

providing a low conductivity, zwitterionic buffer on said device,

electrophoretically transporting said nucleic acid towards a microlocation,

applying current and voltage to the microlocation to effect transportation, whereby the local pH above the microlocation is below the pH of the buffer at its isoelectric point, whereby hybridization between the nucleic acid and a probe located at the microlocation is enhanced.

2. The method for enhanced transport and hybridization of nucleic acids of claim 1, wherein the low conductivity, zwitterionic buffer is histidine.

3. The method for enhanced transport and hybridization of nucleic acids of claim 1, wherein the low conductivity, zwitterionic buffer is L-histidine.

4. The method for enhanced transport and hybridization of nucleic acids of claim 1, wherein the low conductivity, zwitterionic buffer is D-histidine.

5. A method for the effective transport and hybridization of DNA on an active, electronic matrix device, the device having a plurality of microlocations, at least certain of the microlocations including probes, comprising the steps of:

providing a first low-conductivity, zwitterionic buffer to the device,

providing said nucleic acids to the device in said low conductivity, zwitterionic buffer,

applying current and potential to at least certain microlocations so as to effect transport of said nucleic acids to selected microlocations,

changing the buffer to a second buffer with a high salt concentration, and

effecting hybridization of said nucleic acid with said probe at selected microlocations.

6. The method for effecting transport and hybridization of nucleic acids of claim 5, wherein the low conductivity, zwitterionic buffer is cysteine.

7. The method for effecting transport and hybridization of nucleic acids of claim 5, wherein the low conductivity, zwitterionic buffer is alanine.

8. The method for effecting transport and hybridization of nucleic acids of claim 5, wherein the salt concentration is from approximately 50 mM to 100 mM.

9. A method for detection of point mutations in double stranded amplicons comprising the steps of:

providing amplicon products to an active, programmable electronic matrix device,

dilute said products in a low conductance histidine buffer,

denature said products,

hybridize said denatured products in the histidine buffer on the device,

perform stringency so as to discriminate matches versus mismatches, and

detect and analyze said products.

10. The method of claim 9 for detecting point mutations in amplicons wherein the stringency includes electronic stringency.

11. The method of claim 9 for detecting point mutations in amplicons wherein the detection is fluorescent detection.

12. The method of claim 9 for detecting point mutations in amplicons wherein a fluorescent reporter probe sequence is hybridized with said product.