Methods for determination of single nucleic acid polymorphisms using a bioelectronic microchip

• US 7,582,421 B2
• Filed: 09/16/2002
• Issued: 09/01/2009
• Est. Priority Date: 11/01/1993
• Status: Expired due to Fees

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Wang, Ling, Nerenberg, Michael I., Canter, David M., Sosnowski, Ronald G., O'Connell, James P., Radtkey, Ray R.
• Primary Examiner(s): Lu; Frank W
• Application Number:
  US10/245,206
• Publication Number:
  US 20030073122A1
• Time in Patent Office: 2,542 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 422/50, 422/68.1
• 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 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• 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 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 : Electrodes
• 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: 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 1/6827 : for detection of mutation or polymorphism
• 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 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2537/162 : Helper probe
• CPC Class Codes: C12Q 2565/501 : being an array of oligonucleotides
• 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: C12Q 2600/156 : Polymorphic or mutational markers
• 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 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 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00
• CPC Class Codes: Y02A 90/10 : Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

This application includes methods for detecting single nucleotide polymorphisms (SNPs) in a sample using an electronically addressable microchip having a plurality of test sites. A sample nucleic acid is electronically biased, concentrated at, and immobilized to a test site on the microchip. A mixture comprising a first labeled probe and a second labeled probe is electronically hybridized to the sample nucleic acid to form first or second hybridized complexes. The first labeled probe is perfectly complementary to the first sample nucleic acid and the second labeled probe is complementary to the sample nucleic acid and contains a nucleotide that forms a mismatch with the nucleotide at the site of the polymorphism. The first or second hybridized complexes are detected by determining a signal intensity of the label of the first or second probe.

Claims

1. A method for detecting a single nucleotide polymorphism in a sample nucleic acid using an electronically addressable microchip having a plurality of test sites, wherein each of the test sites comprises an individually controllable electrode covered by a permeation layer, the method comprising:

providing a sample nucleic acid suspected of containing a single nucleotide polymorphism;

electronically biasing the sample nucleic acid to a test site of the plurality of test sites on the microchip, and concentrating the sample nucleic acid at the test site;

immobilizing the sample nucleic acid onto the test site;

electronically hybridizing a mixture comprising a first probe with a label selected from the group consisting of fluorescent label, colorimetric label, and chemiluminescent label, and a second probe with a label selected from the group consisting of fluorescent label, colorimetric label, and chemiluminescent label to the sample nucleic acid , wherein the first probe is perfectly complementary to the sample nucleic acid and contains a nucleotide perfectly complementary to the nucleotide at the site of the polymorphism, and forms a first hybridized complex, and the second probe is complementary to the sample nucleic acid and contains a nucleotide which forms a mismatch with the nucleotide at the site of the polymorphism, and forms a second hybridized complex, wherein the label of the first probe and the label of the second probe are different;

subjecting the first and second hybridized complexes to destabilizing conditions sufficient to cause the first probe to dissociate from the first hybridized complex or the second probe to dissociate from the second hybridized complex if there is at least one base-pair mismatch between the first probe or the second probe and the sample nucleic acid; and

detecting the first or second hybridized complex following the subjecting step by determining a signal intensity from the label of the first probe of the first hybridized complex or a signal intensity from the label of the second probe of the second hybridized complex, wherein the single nucleotide polymorphism in the sample nucleic acid is detected if the signal intensity from the label of the first probe of the first hybridized complex is present.

2. The method of claim 1, further comprising the step of subjecting the sample nucleic acid to an amplification reaction before said biasing step.

3. The method of claim 1, wherein the sample nucleic acid further comprises a biotin moiety, and wherein the sample nucleic acid is immobilized onto the test site by a biotin-streptavidin interaction.

4. The method of claim 1, wherein the label of the first probe and the label of the second probe are different fluorophores.

5. The method of claim 4, wherein the different fluorophores are selected from the group consisting of Cy3 and Cy5.

6. The method of claim 1, further comprising the steps of:

providing another sample nucleic acid containing the single nucleotide polymorphism;

electronically biasing the another sample nucleic acid to an another test site of the plurality of test sites on the microchip, concentrating the another sample nucleic acid at the another test site; and

immobilizing the another sample nucleic acid onto the another test site.

7. The method of claim 1, wherein the hybridization step is repeated with at least one additional mixture of the first and second probes.

8. The method of claim 1, wherein the hybridization step is performed under electronic stringency conditions.