Methods for determination of single nucleic acid polymorphisms using a bioelectronic microchip
First Claim
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.
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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.
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Citations
34 Claims
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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:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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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:
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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. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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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:
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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. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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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:
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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.
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Specification