Denaturing high-performance liquid chromatography.
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Chromatography, High Pressure Liquid↗
Biomedical subjects
Andreas Premstaller
Publications and source records attributed to Andreas Premstaller.
Capillary array high-performance liquid chromatography of nucleic acids and proteins.
An array of monolithic poly(styrene/divinylbenzene) capillaries with individual column thermostats was constructed to demonstrate its utility for the separation of nucleic acids, proteins, and tryptic digests in combination with UV absorbance detection. Because of polymerization-related variation in surface area of monolithic columns, the concentration of acetonitrile required for elution of DNA fragments in denaturing HPLC may vary sufficiently to affect the degree of denaturation. Modulation of column temperature offers a convenient way to harmonize elution profiles among columns. Individual regulation of column temperature also provides the means to determine rapidly in a single parallel run the optimum temperature for resolution of biomolecules. Given the high reproducibility of separations among columns and the ease with which poly(styrene/divinylbenzene)-based stationary phases can be modified to accommodate different modes of chromatography, such arrays will find broad applicability in proteogenomics.
Chromatography, High Pressure Liquid↗
Multiplex analysis of single-nucleotide extension products on a 16-capillary, denaturing, high-performance liquid chromatography array.
We constructed a high-performance liquid chromatography array consisting of 16 monolithic poly(styrene/divinylbenzene) capillaries for the parallel multiplex analysis of fluorescent dye-labeled single-nucleotide extension products. Because of the high chemical and physical robustness of the column bed that is covalently linked to the inner surface of the fused silica capillary, the array can be reused thousands of times without replenishment. The choice of fluorophore exerts a significant effect on resolution of the extension products. FAM, HEX, and TAMRA allowed complete resolution of all four possible allelic extension products not only from the extension primer but also from each other. The quantitative accuracy of the method enables the genetic typing of bi- and triallelic single-nucleotide polymorphisms in polyploid genomes and pooled samples.
Chromatography, High Pressure Liquid↗
Genotyping of Snps in a polyploid genome by pyrosequencing.
Single-nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations, and they have become increasingly popular markers for association studies. Allelic discrimination of the mostly binary SNPs has been reported for diploid species, mainly the human, but not for polyploid genomes such as the agriculturally important crops. In the present study, we analyzed the applicability of pyrosequencing to genotyping SNPs in tetraploid potatoes. Out of 94 polymorphic loci tested, 76 (81%) proved to be amenable to allelic discrimination by pyrosequencing. An additional locus could be genotyped by the addition of an ssDNA binding protein to the pyrosequencing reaction. Of the remaining 17 loci, two failed because of the presence of paralogs in the genome, while in the other cases, self-annealing of the primer or template at the low reaction temperature (28 degrees C) employed in pyrosequencing rendered allelic discrimination impossible. The quantitative precision ofpyrosequencing was found to be similar to that of conventional dideoxy sequencing and single-nucleotide primer extension. Exceptfor some sequencespecific limitations, pyrosequencing appears to be an appropriate method for genotying SNPs in polyploid species because it is possible to distinguish not only between homoand heterozygosity but also between the different heterozygous states.
Apyrase↗