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 to produce an amplified mixture of nucleic acid-target regions containing polymorphic loci wherein the amplification conditions comprise between about 400-700 μM of each dNTP and a free Mg2+ concentration between 1.0-2.0 mM, wherein the free Mg2+ concentration is equal to total Mg2+ concentration less total dNTP concentration for all dNTPs;
• (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 reactions 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 thermocycles;
• (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.