HIGH-PRECISION GENOTYPING BY DENATURING CAPILLARY ELECTROPHORESIS

Genotyping, as applied to linkage mapping, human identification, or ma pping of genetic traits, mandates electrophoretic separation systems t hat enable a user to identify alleles with high precision to obtain a correct genotype. For 2-bp microsatellites or short tandem repeats (ST Rs), standard deviations of +/-0.3 nucleotide are required to ensure w ith 99.7% probability the identity or dissimilarity of tested alleles. A complete system, consisting of commercially available laser-induced fluorescence capillary electrophoresis (ABI PRISM 310) and performanc e optimized polymer 4 (POP-4), was evaluated for microsatellite separa tions. POP-4 is a low viscosity polymer for use in uncoated Fused micr obore silica capillaries. It separates DNA fragments that differ in si ze by 1 nucleotide up to 250 nucleotides and that differ in size by 2 nucleotides for fragments up to at least 350 nucleotides in length in about 30 min. The presence of denaturants and, more importantly, opera tion at 60 degrees C was mandatory for high-precision and high-resolut ion sizing operation. Reproducible separation performance was achieved in excess of 100 injections per capillary with resulting standard dev iations in the range of 0.04 to 0.17 nucleotide. Comparative sizing of known CEPH (Centre d'Etudes du Polymorphisme Humaine) samples perform ed at 22 independent test sites showed the usefulness of the system fo r genotyping with standard deviations of 0.24 nucleotide, or better.