Microarray method of genotyping multiple samples at multiple LOCI
First Claim
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1. A method of simultaneously genotyping multiple samples in a single round of hybridization, the method comprising:
- 1) incubating a microarray of polynucleotide samples from multiple individuals with a single solution of a probe mixture of oligonucleotides of known sequence, wherein a) the microarray contains a plurality of samples containing genotypes of interest with each sample in a distinct location, each location occupying an area smaller than or about 1 millimeter across, b) each sample has amplified polynucleotides with a defined segment containing a marker selected from a marker for a gene and markers for allelic variants of the gene, c) the oligonucleotides in the probe mixture are of known sequence and length and have sequences specifically complementary to polynucleotide sequences within the defined segments for each sample for which a genotype is to be determined, wherein the oligonucleotides complementary to the polynucleotides are selected from the group consisting of oligonucleotides with sequences complementary to a segment containing the marker for (1) a gene, (2) one or more allelic variants of the gene, and (3) a gene and one or more allelic variants of the gene, and d) the incubating forms hybrids of polynucleotides of the microarray and complementary oligonucleotides and allows discrimination at single nucleotide resolution; and
2) detecting at the distinct locations on the microarray after a single round of hybridization, stable hybrids formed during the incubation, wherein a hybridization signal indicating the formation of a hybrid or lack of formation of a hybrid genotypes the individuals.
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Abstract
A method for genotyping multiple samples at multiple genetic loci in a single assay is provided. Microarrays of genomic segments representing discrete loci are formed and hybridized with mixtures of synthetic oligonucleotides that are complementary to the genomic segments. Genotyping information is derived by reading the microarray signals. The method can be used to characterize samples from diverse biological sources and for a variety of applications.
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19 Claims
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1. A method of simultaneously genotyping multiple samples in a single round of hybridization, the method comprising:
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1) incubating a microarray of polynucleotide samples from multiple individuals with a single solution of a probe mixture of oligonucleotides of known sequence, wherein a) the microarray contains a plurality of samples containing genotypes of interest with each sample in a distinct location, each location occupying an area smaller than or about 1 millimeter across, b) each sample has amplified polynucleotides with a defined segment containing a marker selected from a marker for a gene and markers for allelic variants of the gene, c) the oligonucleotides in the probe mixture are of known sequence and length and have sequences specifically complementary to polynucleotide sequences within the defined segments for each sample for which a genotype is to be determined, wherein the oligonucleotides complementary to the polynucleotides are selected from the group consisting of oligonucleotides with sequences complementary to a segment containing the marker for (1) a gene, (2) one or more allelic variants of the gene, and (3) a gene and one or more allelic variants of the gene, and d) the incubating forms hybrids of polynucleotides of the microarray and complementary oligonucleotides and allows discrimination at single nucleotide resolution; and
2) detecting at the distinct locations on the microarray after a single round of hybridization, stable hybrids formed during the incubation, wherein a hybridization signal indicating the formation of a hybrid or lack of formation of a hybrid genotypes the individuals. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method of simultaneously genotyping multiple samples in a single round of hybridization, the method comprising:
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1) incubating a microarray of polynucleotide samples from multiple individuals with a single solution of a probe mixture of oligonucleotides of known sequence, wherein a) the microarray contains a plurality of samples containing genotypes of interest with each sample in a distinct location, wherein the microarray contains at least 60 sample locations per cm2, b) each sample has amplified polynucleotides with a defined segment containing a marker selected from a marker for a gene and markers for allelic variants of the gene, c) the oligonucleotides in the probe mixture are of known sequence and length and have sequences specifically complementary to polynucleotide sequences within the defined segments for each sample for which a genotype is to be determined, wherein the oligonucleotides complementary to the polynucleotides are selected from the group consisting of oligonucleotides with sequences complementary to a segment containing the marker for (1) a gene, (2) one or more alielic variants of the gene, and (3) a gene and one or more allelic variants of the gene, and d) the incubating forms hybrids of polynucleotides of the microarray and complementary oligonucleotides and allows discrimination at single nucleotide resolution; and
2) detecting at the distinct locations on the microarray after a single round of hybridization, stable hybrids formed during the incubation, wherein a hybridization signal indicating the formation of a hybrid or lack of formation of a hybrid genotypes the individuals.
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Specification