Patent ID: 6855500

Claim:
A method for genotyping a diploid organism, comprising: providing two alleles of a target gene of a diploid organism suspected to contain a polymorphism; obtaining a first segment of one allele wherein the segment includes the suspected polymorphic locus; obtaining a second segment from the other allele wherein the segment also includes the suspected polymorphic locus; replacing a first natural nucleotide, suspected to be at the polymorphic locus, at greater than 90% of its points of occurrence in the first and the second segment with a first modified nucleotide to give a first and a second modified segment; replacing a second natural nucleotide, suspected to be at the polymorphic locus, at greater than 90% of its points of occurrence in the first and the second modified segments with a second modified nucleotide to give a first and a second twice-modified segment, wherein: replacing the natural nucleotides with modified nucleotides comprises amplification using primers that hybridize to each segment or to each modified segment such that the suspected polymorphic locus will be the first occurrence of a modified nucleotide after the 3′ end of the primer in each amplified segment; covalently bonding a first fluorophore that emits light at a first wavelength to the first modified nucleotide or a residue thereof; covalently bonding a second fluorophore that emits light at a second wavelength to the second modified nucleotide or a residue thereof; cleaving the first and second twice-modified segments at greater than 90% of the points of occurrence of each modified nucleotide to give a first and second set of fragments, one fragment of each segment comprising primer and a modified nucleotide or residue thereof; isolating the two primer-containing fragments; analyzing the isolated fragments for the emission of the first wavelength of light, the second wavelength of light, or both, wherein: if only one of the wavelengths is detected, then the same modified nucleotide was incorporated at the suspected polymorphic locus in both the first and the second allelic segments indicating that the alleles of the target gene are homozygous, whereas if both wavelengths are detected, then a different modified nucleotide was incorporated at the suspected polymorphic locus in the first and the second allelic segments indicating that the alleles of the target gene are heterozygous.