Patent ID: 8349566

Claim:
A multiplex method of genotyping a plurality of polymorphic loci, comprising: (a) simultaneously amplifying a plurality of nucleic acid-target regions with amplification primer pairs under amplification conditions to produce an amplified mixture of nucleic acid-target regions containing polymorphic loci wherein the amplification conditions comprise dNTPs and for at least one amplification primer pair, one primer is in lower concentration than the other primer; (b) after (a) contacting the amplified mixture of nucleic acid-target regions with genotyping primers in the presence of at least one chain terminating reagent under primer mass extension conditions whereby the primers are extended up to, or through, the respective polymorphic loci, wherein there is one genotyping primer for each polymorphic locus and wherein the extension conditions comprise at least 55 thermal cycles; (c) determining the mass of the extended genotyping primers; and (d) determining the genotypes of the plurality of polymorphic loci from the mass of the extended genotyping primers.