Polymorphism detection
First Claim
1. A method of identifying whether a target nucleic acid sequence includes a polymorphism, comprising:
- hybridizing said target nucleic acid sequence to an array comprising at least one detection block of probes, said detection block including a first group of probes that are complementary to said target nucleic acid sequence except that the first group of probes includes a plurality of monosubstitutions of positions in said sequence that are within n bases of a base in said sequence that is complementary to said polymorphism, wherein n is from 0 to 5, and a second and third group of probes complementary to marker-specific regions upstream and downstream of the target nucleic acid sequence, wherein the third group of probes differs from the second set of probes at single bases corresponding to known mismatch positions; and
determining hybridization intensities of the target nucleic acid and the marker-specific regions to identify said polymorphism.
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Abstract
The present invention generally provides a rapid efficient method for analyzing polymorphic or biallelic markers, and arrays for carrying out these analyses. In general, the methods of the present invention employ arrays of oligonucleotide probes that are complementary to target nucleic acids which correspond to the marker sequences of an individual. The probes are typically arranged in detection blocks, each block being capable of discriminating the three genotypes for a given marker, e.g., the heterozygote or either of the two homozygotes. The method allows for rapid, automatable analysis of genetic linkage to even complex polygenic traits.
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Citations
20 Claims
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1. A method of identifying whether a target nucleic acid sequence includes a polymorphism, comprising:
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hybridizing said target nucleic acid sequence to an array comprising at least one detection block of probes, said detection block including a first group of probes that are complementary to said target nucleic acid sequence except that the first group of probes includes a plurality of monosubstitutions of positions in said sequence that are within n bases of a base in said sequence that is complementary to said polymorphism, wherein n is from 0 to 5, and a second and third group of probes complementary to marker-specific regions upstream and downstream of the target nucleic acid sequence, wherein the third group of probes differs from the second set of probes at single bases corresponding to known mismatch positions; and
determining hybridization intensities of the target nucleic acid and the marker-specific regions to identify said polymorphism. - View Dependent Claims (2, 3, 6, 7, 8, 9, 10)
a) calculating the control difference between the average of the hybridization intensities of the second group of probes, the hybridization intensities comprising control perfect matches (PM), minus the average of the hybridization intensities, the hybridization intensities comprising control single-base mismatches (MN);
b) calculating the possible perfect match intensity and a heteromismatch intensity from the hybridization intensities for each position of monosubstitutions of the first group of probes;
c) calculating the difference between the possible perfect match intensity and the heteromismatch intensity for each position of monosubstitutions of the first group of probes;
d) calculating a normalized difference (ND) by dividing the difference of step c) by control difference; and
e) using principal component analysis, identifying a polymorphism by comparing normalized differences between individuals in a population.
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9. The method of claim 8, wherein an ND is calculated for at least two alleles of a polymorphism.
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10. The method of claim 9, wherein homozygotes and heterozygotes are detected by combining principal component analysis for the at least two alleles.
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4. A method of identifying whether a target nucleic acid sequence includes a polymorphism, comprising:
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hybridizing said target nucleic acid sequence to an array comprising at least one detection block of probes, said detection block including a first group of probes that are complementary to said target nucleic acid sequence except that the first group of probes includes a plurality of monosubstitutions of positions in said sequence that are within n bases of a base in said sequence that is complementary to said polymorphism, wherein n is from 0 to 5, and a second and third group of probes complementary to marker-specific regions upstream and downstream of the target nucleic acid sequence, wherein the third group of probes differs from the second set of probes at single bases corresponding to known mismatch positions, wherein said target nucleic acid comprises a detectable label that is a binding group; and
determining hybridization intensities of the target nucleic acid and the marker-specific regions to identify said polymorphism. - View Dependent Claims (5)
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11. A method of determining a genotype of a sample of nucleic acid sequences, comprising:
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receiving a first hybridization intensity indicating hybridization affinity between a first set of probes and a first allele, the first set of probes being perfectly complementary to a portion of the first allele;
receiving a second hybridization intensity indicating hybridization affinity between a second set of probes and the first allele, the second set of probes having a single base mismatch with a portion of the first allele;
calculating a first difference between the first and second hybridization intensities;
receiving a third hybridization intensity indicating hybridization affinity between a third set of probes and a second allele, the third set of probes being perfectly complementary to a portion of the second allele;
receiving a fourth hybridization intensity indicating hybridization affinity between a fourth set of probes and the second allele, the fourth set of probes having a single base mismatch with a portion of the second allele;
calculating a second difference between the third and fourth hybridization intensities; and
comparing the first and second differences in order to determine the genotype of the sample of nucleic acid sequences. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification