Patent ID: 8153363
Filing Date: 2012-04-10
Classification: C12Q,G16B

Abstract:
1. An in vitro method for genotyping genetic variations in an individual, the method comprising: (a) providing a sample containing nucleic acid which comprises the genetic variations to be genotyped (the target DNA), (b) providing, for each genetic variation to be genotyped, oligonucleotide probe pairs, wherein, (c) amplifying and detectably labelling the target DNA; (d) contacting the target DNA with the probes under conditions which allow hybridization to occur, thereby forming detectably labeled nucleic acid-probe hybridization complexes, (e) determining the intensity of detectable label for each probe, thereby obtaining a raw intensity value for each particle type; (f) optionally amending the raw intensity value to take account of background noise, thereby obtaining a clean intensity value for each replica; and (g) applying an algorithm to the intensity data from (e) or (f), thereby determining the genotype with respect to each genetic variation, wherein application of the algorithm comprises calculating a raw intensity value from the intensity values for each of the replicas of each probe coupled with a particle, and wherein the algorithm comprises deriving: a first linear function: a1ratio1+b1ratio2+c1 that characterizes genotype AA: a second linear function: a2ratio1+b2ratio2+c2 that characterizes genotype AB; and a third linear function: a3ratio1+b3ratio2+c3 that characterizes genotype BB; wherein: a1 is the coefficient which accompanies the X in the linear function for the genotype AA; a1 being obtained by applying a discriminate analysis to the ratios 1 and 2 obtained from analysing Z patients for the genotype AA, Z for BB and Z for AB, wherein Z is a number more than two, b1 is the coefficient which accompanies the Y in the linear function for the genotype AA; b1 being obtained by applying a discriminate analysis to the ratios 1 and 2 obtained from analysing Z patients for the genotype AA, Z for BB and Z for AB; c1 is the independent term of the first linear function; a2 is the coefficient which accompanies the X in the linear function for the genotype AB; a2 being obtained by applying a discriminate analysis to the ratios 1 and 2 obtained from analysing Z patients for the genotype AA, Z for BB and Z for AB; b2 is the coefficient which accompanies the Y in the linear function for the genotype AB; b2 being obtained by applying a discriminate analysis to the ratios 1 and 2 obtained from analysing Z patients for the genotype AA, Z for BB and Z for AB; c2 is the independent term of the second linear function; a3 is the coefficient which accompanies the X in the linear function for the genotype BB; a3 being obtained by applying a discriminate analysis to the ratios 1 and 2 obtained from analysing Z patients for the AA, Z for BB and Z for AB; b3 is the coefficient which accompanies the Y in the linear function for the genotype BB; b3 being obtained by applying the discriminate analysis to the ratios 1 and 2 obtained from analysing Z patients for the genotype AA, Z for BB and Z for AB; c3 is the independent term of the third linear function; one probe is used for each allele, comprising probes 1 and 2 (oligo 1 and oligo 2), wherein probe 1 corresponds to allele one and probe 2 corresponds to allele two; ratio 1 is the proportion of the median of the intensities of the particles of the same type linked to oligo 1 which detects the allele one divided by the median of the intensities of the oligo 1 plus the median of the intensities of intensities of the particles of the same type linked to oligo 2 and can be calculated by the equation: and ratio 2 is the proportion of the median of the intensities of the particles of the same type linked to oligo 2 which detects the allele two divided by the median of the intensities of the oligo 1 plus the median of the intensities of intensities of the particles of the same type linked to oligo 2 and can be calculated by the equation: