Hans Erik Söderlund

Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequencing : application to aspartylglucosaminuria in Finland

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal disease caused by inadequate aspartylglucosaminidase (AGA) activity. The disease is prevalent in the genetically isolated Finnish population. We have used a new method, solid-phase minisequencing, to determine the frequency of two missense mutations in the AGA gene in this population. In samples from 70% of the Finnish AGU families, we found that the two nucleotide changes were always associated, and they were identified in 98% of the AGU alleles analyzed. Thus, the high prevalence of AGU in the Finnish population is the consequence of a founder effect of one ancient mutation. The identification of asymptomatic carriers by the minisequencing test proved to be unequivocal. The method also allowed quantification of a mutated nucleotide sequence present in less than 1% of a sample. The frequency of AGU carriers in this population was 1/36 when estimated by quantifying the mutated AGU allele in a pooled leukocyte sample from 1350 normal Finnish individuals.

Sensitive detection of genes by sandwich hybridization and time-resolved fluorometry

Europium has been used as a non-radioactive marker in immunoassays as this metal can be detected with high sensitivity by time-resolved fluorometry. In this work streptavidin labeled with europium was used to detect biotinylated probes in a sandwich nucleic-acid hybridization assay with microtitration strips as the solid phase. pBR 322 plasmids were detected with a sensitivity of 4 x 10(5) molecules. As the sample is added in solution in sandwich hybridization, fast and simple sample pre-treatment can be used without encountering background problems. The method was applied to test bacterial samples of uropathogenic Escherichia coli strains for the presence of the beta-lactamase gene.

Nucleic acid sandwich hybridization: enhanced reaction rate with magnetic microparticles as carriers

A method for the detection of nucleic acid hybrids using the sandwich hybridization technique with magnetic polystyrene microparticles as the solid support is described. The capture DNA is coupled to the polystyrene-hydroxy surface of the particles through p-toluenesulfonyl chloride activation. The use of microparticles results in a substantial increase in the reaction rate compared to filter hybridization, without decreasing the sensitivity of detection. Polyethylene glycol additionally enhances the reaction rate. The use of magnetic microparticles allows rapid and convenient collection of the formed hybrids.