David M. Demorest

Genotyping, as applied to linkage mapping, human identification, or mapping of genetic traits, mandates electrophoretic separation systems that enable a user to identify alleles with high precision to obtain a correct genotype. For 2-bp microsatellites or short tandem repeats (STRs), standard deviations of +/-0.3 nucleotide are required to ensure with 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 performance optimized polymer 4 (POP-4), was evaluated for microsatellite separations. POP-4 is a low viscosity polymer for use in uncoated fused microbore silica capillaries. It separates DNA fragments that differ in size 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, operation at 60 degrees C was mandatory for high-precision and high-resolution sizing operation. Reproducible separation performance was achieved in excess of 100 injections per capillary with resulting standard deviations in the range of 0.04 to 0.17 nucleotide. Comparative sizing of known CEPH (Centre d'Etudes du Polymorphisme Humaine) samples performed at 22 independent test sites showed the usefulness of the system for genotyping with standard deviations of 0.24 nucleotide, or better.

Obtaining accurate molecular weight estimates for glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moieties of the proteins. This leads to lower charge-to-mass ratios for SDS-glycoprotein complexes, resulting in over-estimation of molecular weights by SDS-PAGE. In order to minimize these inaccuracies for proteins with abnormal charge-to-mass ratios, a Ferguson plot may be employed. This application requires the determination of relative mobilities for standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular because it requires time-consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates all of the data required for a Ferguson plot using a replaceable sieving matrix (thereby eliminating gel polymerization) in a capillary format. In addition, this technique possesses the advantages inherent to capillary electrophoresis, namely, very fast separation times, and on-line monitoring which allows quantitation and precludes post-separation staining and destaining of gels.