Patent ID: 6479244
Filing Date: 2002-11-12
Classification: C12Q

Abstract:
A method for genotyping different alleles of a microsatellite DNA locus said microsatellite DNA locus having a repeat region and further having a predetermined number of different alleles, each allele having a different number of microsatellite repeat units in said repeat region, said method comprising:(a) providing a sample containing the microsatellite DNA locus; (b) providing a set of oligonucleotides comprising: (i) a 5â€² primer complementary to a 3â€² flanking region of the repeat region of the microsatellite DNA locus; (ii) a central primer comprising a number of primer repeat units complementary to the repeat region corresponding to the allele having the least number of said microsatellite repeat units; and, (iii) an ensemble of discriminating 3â€² primers comprising a plurality of 3â€² primers each of which has the formula of XYn, wherein X is a sequence that is complementary to the a 5â€² flanking sequence of the repeat region of the microsatellite DNA Y is a primer repeat unit located at the 5â€² end of each of said 3â€² primers which contains said primer repeat unit, the primer repect unit complementary to the microsatellite repeat unit; and, n is the number of primer repeat units in any one of said discriminating 3â€² primers wherein n is a varying number selected such that a combination of oligonucleotides comprising said 5â€² primer, said central primer and any one of said discriminating 3â€² primers is complementary to one of said predetermined number of different alleles of the DNA microsatellite locus; (c) mixing the sample and primer such that the primers and microsatellite DNA hybridize; (d) adding a ligating reagent; (e) detecting the presence of a ligation product that incorporates three oligonucleotides, said three oligonucleotides comprising said 5â€² primer, said central primer and one of said discriminating 3â€² primers; and (f) deducing the genotype of the sample according to the identities of the oligonucleotides incorporated in a ligation product.