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

• US 20030073122A1
• Filed: 09/16/2002
• Published: 04/17/2003
• Est. Priority Date: 11/01/1993
• Status: Active Grant

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: OʼConnell, James P., Wang, Ling, Sosnowski, Ronald G., Nerenberg, Michael I., Canter, David M., Radtkey, Ray R.
• Granted Patent: US 7,582,421 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

Methods are provided for the analysis and determination of the nature of single nucleic acid polymorphisms (SNPs) in a genetic target. In one method of this invention, the nature of the SNPs in the genetic target is determined by the steps of providing a plurality of hybridization complexes arrayed on a plurality of test sites on an electronically bioactive microchip, where the hybridization complex includes at least a nucleic acid target containing a SNP, a stabilizer probe having a sequence complementary to the target sequence and/or reporter probe, and a reporter probe having a selected sequence complementary to either the stabilizer or the same target sequence strand wherein a selected sequence of the reporter includes either a wild type nucleotide or a nucleotide corresponding to the SNP of the target. In accordance with the invention, the stabilizer, reporter and target amplicons are hybridized using electronic assistance of the microchip system such that base-stacking energies are utilized in discerning among other identifying indicators, the presence of wild type or polymorphism sequence. Applications include disease diagnostics, such as for the identification of polymorphisms in structural genes, regulatory regions, antibiotic or chemotherapeutic resistance conferring regions, or for SNPs associated with speciation or used for determination of genetic linkage.

Claims

1. A method for determining single nucleic acid polymorphisms in target nucleic acids of interest using base-stacking energies and an electronically addressable microchip comprising:

a. providing target nucleic acids of interest that are or are not subjected to an amplification reaction using either at least one amplification primer for linear amplification or at least one pair of amplification primers for exponential amplification to form amplification products of said targets;

b. electronically addressing said target nucleic acids or said amplification products to at least one capture site on said microchip while in the presence of first sets of “

stabilizer”

oligonucleotides that are complementary to at least one nucleic acid strand of said target nucleic acids or said amplification products, said stabilizer oligonucleotides and the target or amplification product strand to which said stabilizer oligonucleotides are complementary further forming hybridized complexes;

c. capturing said hybridized complexes to said capture site;

d. hybridizing to said complexes second sets of “

reporter”

oligonucleotides which are complementary to the same strand of said target nucleic acids or amplification product to which said stabilizer is complementary to form ternary structures, said reporter oligonucleotides further hybridizing to said complex such that said stabilizer and said reporter oligonucleotides are annealed to said target nucleic acids or amplification products at positions adjacent to one another;

c. subjecting said ternary structures to destabilizing conditions sufficient to cause said reporters to dissociate from said ternary structures if there is at least one base-pair mismatch between said reporters and said target nucleic acids or amplification products, and

f detecting a loss or a retention of said reporters from said capture site.

2. A method according to claim 1 wherein said amplification reaction is carried out using an amplification method selected from the group consisting of PCR, SDA, NASBA, TMA, rolling circle, T7, T3, and SP6.

3. A method according to claim 1 wherein interfering oligonucleotides are included in step (b) of claim 1, said interfering oligonucleotides having sequence complementary to a target nucleic acids or amplification product strand that is not the same strand to which said stabilizer oligonucleotide is complementary.

4. A method according to claim 1 wherein at least one of the primers used in said amplification reaction is biotinylated.

5. A method according to claim 4 wherein an amplicon strand derived from a biotinylated primer becomes anchored by its biotin moiety to said capture site in step (c) of claim 1.

6. A method according to claim 1 wherein said stabilizer oligonucleotide is biotinylated.

7. A method according to claim 6 wherein said biotinylated stabilizer oligonucleotide is anchored by its biotin moiety to said capture site in step.

8. A method according to claim 1 wherein said reporter oligonuclgotides are labeled using a label selected from the group consisting of fluorescin derivatives, bodipy derivatives, Rhodamine derivatives, and Cy dyes

9. A method according to claim 1 wherein said destabilizing conditions are created by methods selected from the group consisting of performing electronic biasing, making temperature adjustments, making ionic strength adjustments, and making adjustments in pH.

10. A method according to claim 1 wherein said reporter oligonucleotides have as their 3′

, 5′

or both 3′ and

5′

terminal bases, a nucleic acid base complementary to either a wild type base or a mutation base found in said target nucleic acids.

11. A method according to claim 1 wherein a multiplicity of said target nucleic acids or amplification products that form said complexes are electronically biased to a single capture site.

12. A method for determining single nucleic acid polymorphisms in target nucleic acids of interest using base-stacking energies and an electronically addressable microchip comprising:

a. providing nucleic acid from two spaced apart regions of target nucleic acids of interest that are or are not subjected to an amplification reaction using at least one amplification primer for linear amplification or at least one pair of amplification primers for exponential amplification for each said regions to form amplification products of said regions, said regions further comprising target nucleic acid sequences that are (i) located 3′

or 5′

to sequence in said target susceptible of containing single nucleic acid polymorphisms and/or (ii) are susceptible of containing target-specific nucleic acid information;

b. electronically addressing said regions of target nucleic acids or amplification products thereof to at least one capture site on said microchip while in the presence of first sets of “

stabilizer”

oligonucleotides that contain nucleic acid sequences that are complementary to said regions of the target nucleic acids or amplification products thereof, said stabilizer oligonucleotides further comprising nucleic acid sequences that span at least one terminal portion of each of said spaced apart regions of said target nucleic acids or amplification products thereof and further comprise nucleic acid sequences complementary to said target along a region of the target located between said spaced apart regions, said target nucleic acids or said amplification products thereofand stabilizer oligonucleotides further forming a ternary hybridized complex;

c. capturing said ternary hybridized complex to said capture site;

d. hybridizing to said ternary complex at least one second sets of “

reporter”

oligonucleotides which are complementary to a portion or to all of the stabilizer oligonucleotide along that section of said stabilizer lying between the portions of the stabilizer that are complementary to the two spaced apart regions to form at least a quaternary structure, said reporter oligonucleotides further hybridizing to said complex such that said reporter oligonucleotides are annealed to said stabilizer at positions adjacent to and in between said spaced apart regions;

e. subjecting said quaternary structure to destabilizing conditions sufficient to cause any of said reporters to dissociate from said quaternary structure if there is at least one base-pair mismatch between said reporter and said stabilizer oligonucleotide; and

f. detecting a loss or a retention of said reporters from said capture site.

13. A method according to claim 12 wherein said amplification reaction is carried out using an amplification method selected from the group consisting of PCR, SDA, NASBA, TMA, rolling circle, T7, T3, and SP6.

14. A method according to claim 12 wherein interfering oligonucleotides are included in step (b) of claim 12, said interfering oligonucleotides having sequence complementary to a target nucleic acid or amplification product strand thereof that is not the same strand to which said stabilizer oligonucleotide is complementary.

15. A method according to claim 12 wherein at least one of the primers used in said amplification reaction is biotinylated.

16. A method according to claim 15 wherein an amplicon strand derived from a biotinylated primer becomes anchored by its biotin moiety to said capture site in step (c) of claim 12.

17. A method according to claim 12 wherein said stabilizer oligonucleotide is biotinylated.

18. A method according to claim 17 wherein said biotinylated stabilizer oligonucleotide becomes anchored by its biotin moiety to said capture site.

19. A method according to claim 12 wherein said reporter oligonucleotides are labeled using a label selected from the group consisting of fluorescin derivatives, bodipy derivatives, Rhodamine derivatives, and Cy dyes.

20. A method according to claim 12 wherein said destabilizing conditions are created by methods selected from the group consisting of performing electronic biasing, making temperature adjustments, making ionic strength adjustments, and making adjustments in pH.

21. A method according to claim 12 wherein said reporter oligonucleotides have as their 3′

, 5′

or both 3′ and

5′

terminal bases, a nucleic acid base complementary to either a wild type base found in said target nucleic acids, a mutation base found in said target nucleic acids, or a base in said stabilizer oligonucleotide.

22. A method according to claim 12 wherein a multiplicity of said target nucleic acids or amplification products thereof that form said complexes are electronically biased to a single capture site.

23. A method for determining single nucleic acid polymorphisms in target nucleic acids of interest using base-stacking energies and an electronically addressable microchip comprising:

a. providing nucleic acid sequence from at least two genetic loci of target nucleic acids of interest that are or are not be subjected to an amplification reaction using at least one primer for linear amplification or at least one pair of amplification primers for exponential amplification for each locus to form amplification products of said loci, said loci further comprising target nucleic acid sequences that are (i) located 3′

or 5′

to sequence in said target susceptible of containing single nucleic acid polymorphisms and/or (ii) are susceptible of containing target-specific nucleic acid information;

b. electronically addressing said target loci or amplification products thereof to at least one capture site on said microchip while in the presence of first sets of “

stabilizer”

oligonucleotides that contain nucleic acid sequence that is complementary to said target loci or amplification products thereof, said stabilizer oligonucleotides further comprising nucleic acid sequence that is complementary to five or more 5′

or 3′

terminal region bases of said target loci or amplification products thereof, said stabilizer further comprising nucleic acid sequences complementary to at least one of a second sets of reporter oligonucleotides, said target loci or amplification products thereof and said stabilizer oligonucleotides further forming a ternary hybridized complex;

c. capturing said ternary hybridized complex to said capture site;

d. hybridizing to said ternary complex at least one of said sets of “

reporter”

oligonucleotides to form at least a quaternary structure, said reporter oligonucleotides further hybridizing to said ternary complex such that sequences from each of said target loci or amplification products thereof and said reporter oligonucleotides are annealed to said stabilizer oligonucleotides at positions on said stabilizer adjacent to one another, said reporter oligonucleotides hybridizing to said stabilizer oligonucleotide between said target loci or amplification product sequences;

e. subjecting said quaternary structure to destabilizing conditions sufficient to cause any of said reporters to dissociate from said quaternary structure if there is at least one base-pair mismatch between said reporter and said stabilizer, or between said stabilizer and said target loci or amplification product; and

f. detecting a loss or a retention of said reporters from said capture site.

24. A method according to claim 23 wherein said amplification reaction is carried out using an amplification method selected from the group consisting of PCR, SDA, NASBA, TMA, rolling circle, T7, T3, and SP6.

25. A method according to claim 23 wherein interfering oligonucleotides are included in step (b) of claim 23, said interfering oligonucleotides having sequence complementary to the target loci or amplification product nucleic acid strands that are not the same strands to which said stabilizer oligonucleotide is complementary.

26. A method according to claim 23 wherein at least one of the primers used in said amplification reaction is biotinylated.

27. A method according to claim 26 wherein an amplicon strand derived from a biotinylated primer becomes anchored by its biotin moiety to said capture site in step (c) of claim 23.

28. A method according to claim 23 wherein said stabilizer oligonucleotide is biotinylated.

29. A method according to claim 28 wherein said biotinylated stabilizer oligonucleotide becomes anchored by its biotin moiety to said capture site.

30. A method according to claim 23 wherein said reporter oligonucleotides are labeled using a label selected from the group consisting of fluorescin derivatives, bodipy derivatives, Rhodamine derivatives, and Cy dyes.

31. A method according to claim 23 wherein said destabilizing conditions are created by methods selected from the group consisting of performing electronic biasing, making temperature adjustments, making ionic strength adjustments, and making adjustments in pH.

32. A method according to claim 23 wherein said reporter oligonucleotides have as their 3′

, 5′

or both 3′ and

5′

terminal bases, a nucleic acid base complementary to either a wild type base found in said target loci, a mutation base found in said target loci, or to said stabilizer oligonucleotide.

33. A method according to claim 23 wherein a multiplicity of said target nucleic acids or amplification products thereof that form said complexes are electronically biased to a single capture site.

34. A method for multiplex analysis of single nucleic acid polymorphisms in target nucleic acids of interest using base-stacking energies and an electronically addressable microchip comprising:

a. providing a plurality of samples containing target nucleic acids of interest;

b. performing an amplification reaction on said samples to produce amplification products of said target nucleic acids in said sample;

c. electronically addressing said amplification products to specific capture sites in the presence of stabilizer probe that has complementary sequence to said amplification products to form hybridization complexes between said amplification products and said stabilizer;

d. anchoring said complex to a capture site;

e. simultaneously or sequentially detecting the presence of SNPs in the nucleic acid of said plurality of samples by detecting stabilization of hybridization of a reporter probe that anneals to either said amplification products or to the stabilizer probe, and further anneals either adjacent to said stabilizer probe or adjacent to said amplification products respectively.