Methods for high level multiplexed polymerase chain reactions and homogeneous mass extension reactions

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 whereby at least 60% of 7 or more nucleic acid target regions attempted are amplified by 7 or more amplification primer pairs to produce an amplified mixture of nucleic acid-target regions containing polymorphic loci, wherein the amplification conditions comprise dNTPs and free Mg2+ at concentrations whereby at least 60% of the genotypes for said 7 or more nucleic acid target regions attempted are determined;
• (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 reaction conditions whereby the primers are extended up to, or through, the respective polymorphic loci, wherein there is one genotyping primer for each polymorphic locus;
• (c) determining the masses of the extended genotyping primers;
• and
• (d) based on the masses of the extended genotyping primers determined in (c), determining at least 60% of the genotypes for said 7 or more nucleic acid target regions attempted, thereby genotyping a plurality of polymorphic loci.