Mel N. Kronick

Application of automated DNA sizing technology for genotyping microsatellite loci

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories.

Phycobiliproteins as Labels in Immunoassay

Fluorescence has for many years been viewed as a potential means of providing high sensitivity in a non-isotopic test format (Soini and Hemmila, 1979; Hemmila, 1985). As Jolley et al. (1984) have clearly pointed out, the number of photons emitted from a sample of fluorescently tagged molecules can exceed the number of gamma ray photons emitted from the same number of gamma-ray-emitting tagged molecules. The reason for this is two fold: each radioactive molecule can emit only once and it emits its photon only when it naturally and spontaneously decays. Nevertheless, high sensitivity assays using fluorescence have not replaced radioactive assays to any great extent. Non-isotopic versions of most high sensitivity assays are run today using enzymes as labels. Fluorescence immunoassay has penetrated the marketplace but only in certain applications. One technique, fluorescence polarization (Dandliker and Saussure, 1970; Jolley et al., 1981), has proven very useful, but only for small molecules such as drugs which are present at concentrations from approximately 10−6 to 10−10 M. The dynamic range inherent in fluorescence polarization assays in a competition assay configuration result in this limitation and the physics of fluorescence polarization prevents its being adapted easily to immunometric assays or even to competition assays for larger (≳1000 molecular weight) molecules. Assays based upon fluorescence quenching or fluorescence energy transfer (Ullman et al., 1976; Ullman and Khanna, 1981) similarly have proven most useful to date only in competition assays for analytes of concentration greater than about 10−10 M.

Optimal Size Calling Methods for Electrophoretic Analysis Utilizing Internal Size Standards

A technically demanding problem in analytical genetics is quantitative matching of electrophoretic bands. The number usually associated with a match in forensic science is the size of the nucleic acid molecule in base pairs. Unfortunately, there are many inherent difficulties in the accurate quantitative determination of the size of DNA fragments.