Single nucleotide polymorphic discrimination by electronic dot blot assay on semiconductor microchips
First Claim
1. A method for detecting at least one single nucleotide polymorphism in at least one patient nucleic acid containing sample using an electronically addressable microchip having a plurality of test sites, wherein each test site comprises an individually controllable electrode covered by a permeation layer, the method comprising:
- i. providing at least one sample nucleic acid containing at least one target nucleic acid sequence of interest that contains at least one single nucleotide polymorphism locus;
ii. electronically biasing one or more specified test sites on the microchip in order to concentrate the sample nucleic acid at the specified test sites;
iii. immobilizing the sample nucleic acid onto the test sites;
iv. electronically hybridizing a mixture comprising first and second differently labeled nucleic acid probes to the immobilized sample nucleic acid to form hybridized complexes, wherein the first probe contains a sequence specific for one allele of the single nucleotide polymorphism of the locus, and the second probe contains a sequence differing from the first sequence by a single base;
v. performing electronic stringency on said hybridized complexes to destabilize hybridization complexes comprising at least one mismatch between the immobilized sample nucleic acid and the hybridized probe; and
vi. detecting hybridization complexes that remain following the electronic stringency step (v) by detecting the hybridized labeled probes.
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Abstract
A rapid assay for single nucleotide polymorphism (SNP) detection that utilizes electronic circuitry on silicon microchips is disclosed. The method provides accurate discrimination of amplified DNA samples following electronic assisted transport, concentration, and attachment of DNA to selected electrodes (test sites). The test sites make up organized arrays of samples that are distinguished by using internal controls of dual labeled reporters comprising wild-type and mismatched sequences to validate the SNP genotype. This method has been used to discriminate the complex quadra-allelic SNP of mannose binding protein.
27 Citations
21 Claims
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1. A method for detecting at least one single nucleotide polymorphism in at least one patient nucleic acid containing sample using an electronically addressable microchip having a plurality of test sites, wherein each test site comprises an individually controllable electrode covered by a permeation layer, the method comprising:
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i. providing at least one sample nucleic acid containing at least one target nucleic acid sequence of interest that contains at least one single nucleotide polymorphism locus;
ii. electronically biasing one or more specified test sites on the microchip in order to concentrate the sample nucleic acid at the specified test sites;
iii. immobilizing the sample nucleic acid onto the test sites;
iv. electronically hybridizing a mixture comprising first and second differently labeled nucleic acid probes to the immobilized sample nucleic acid to form hybridized complexes, wherein the first probe contains a sequence specific for one allele of the single nucleotide polymorphism of the locus, and the second probe contains a sequence differing from the first sequence by a single base;
v. performing electronic stringency on said hybridized complexes to destabilize hybridization complexes comprising at least one mismatch between the immobilized sample nucleic acid and the hybridized probe; and
vi. detecting hybridization complexes that remain following the electronic stringency step (v) by detecting the hybridized labeled probes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification