Patent ID: 8119345

Claim:
A method for genotyping in typing assays applied to a biological sample, the sample containing a nucleic acid target of interest, the target nucleic acid being susceptible to having a single nucleotide polymorphism (SNP), comprising the steps of: performing a real-time amplification of the target, generating multiple copies of amplicons, in the presence of at least two different labeled probes, each probe allowing real-time detection at an SNP position of both a wildtype and at least one possible mutation, actively calculating a discriminatory variable value based on signals of each combination of the at least two different labeled probes, and discriminating between the genotypes based on the discriminatory variable value, wherein: when the SNP is a bi-allelic SNP, the discriminatory variable value for a heterozygous genotype is a ratio selected from the group consisting of (k WT→WT +k WT→m )/(k m→m +k m→WT ), (k WT→WT −k WT→m )/(k m→m −k m→WT ), (k WT→WT ×k WT→m )/(k m→m ×k m→WT ), (k WT→WT /k WT→m )/(k m→m /k m→WT ), and their reciprocals; the discriminatory variable value for a homozygous mutant genotype is a ratio of k WT→m /k m→m or its reciprocal; the discriminatory variable value for a homozygous wildtype is a ratio of k WT→WT /k m→WT or its reciprocal; when the SNP is a tri-allelic SNP, the discriminatory variable value for an AAA genotype is a ratio of k A → A /k G → A or its reciprocal; the discriminatory variable value for an AAG genotype is a ratio selected from the group consisting of (2k A → A +k A → G )/(2k G → A +k G → G ), (2k A → A −k A → G )/(2k G → A −k G → G ), (2k A → A ×k A → G )/(2k G → A ×k G → G ), (2k A → A /k A → G )/(2k G → A /k G → G ), and their reciprocals; the discriminatory variable value for an AGG genotype is a ratio selected from the group consisting of (k A → A +2k A → G )/(k G → A +2k G → G ), (k A → A −2k A → G )/(k G → A −2k G → G ), (k A → A ×2k A → G )/(k G → A ×2k G → G ), (k A → A /2k A → G )/(k G → A /2k G → G ), and their reciprocals; and the discriminatory variable value for a GGG genotype is a ratio of k A → G /k G → G or its reciprocal.