Enzymatic on-chip enhancement for high resolution genotyping DNA microarrays.

Antibiotic resistance among pathogenic microorganisms is emerging as a major human healthcare concern. While there are a variety of resistance mechanisms, many can be related to single nucleotide polymorphisms and for which DNA microarrays have been widely deployed in bacterial genotyping. However, genotyping by means of allele-specific hybridization can suffer from the drawback that oligonucleotide probes with different nucleotide composition have varying thermodynamic parameters. This results in unpredictable hybridization behavior of mismatch probes. Consequently, the degree of discrimination between perfect match and mismatch probes is insufficient in some cases. We report here an on-chip enzymatic procedure to improve this discrimination in which false-positive hybrids are selectively digested. We find that the application of CEL1 Surveyor nuclease, a mismatch-specific endonuclease, significantly enhances the discrimination fidelity, as demonstrated here on a microarray for the identification of variants of carbapenem resistant Klebsiella pneumoniae carbapenemases and monitored by end point detection of fluorescence intensity. Further fundamental investigations applying total internal reflection fluorescence detection for kinetic real-time measurements confirmed the enzymatic enhancement for SNP discrimination.

Genotyping DNA chip for the simultaneous assessment of antibiotic resistance and pathogenic potential of extraintestinal pathogenic Escherichia coli.

Urinary tract infections (UTIs) are among the most frequently occurring infections and are mostly caused by extraintestinal pathogenic Escherichia coli. DNA microarrays are potent molecular diagnostic tools for rapid diagnosis of bacterial infections with high relevance for UTIs. In this study, we present the integration and application of two DNA chip modules for the simultaneous detection of single nucleotide polymorphisms in gyrA (quinolone resistance) and fimH (increased adhesion to urinary tract epithelium). The performance of the combined diagnostic chip was assessed by genotyping 140 E. coli strains. Resistance-causing mutations could only be identified in UTI isolates. A complete genotyping assay could be performed in <4h after DNA extraction. Together with the excellent genotyping results, this constitutes a competitive alternative as a standard tool for routine clinical diagnostics.

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriated Saccharomyces cerevisiae.

This study addresses the question of whether observable changes in fluxes in the primary carbon metabolism of Saccharomyces cerevisiae occur between the different phases of the cell division cycle. To detect such changes by metabolic flux analysis, a 13C-labeling experiment was performed with a fed-batch culture inoculated with a partially synchronized cell population obtained through centrifugal elutriation. Such a culture exhibits dynamic changes in the fractions of cells in different cell cycle phases over time. The mass isotopomer distributions of free intracellular metabolites in central carbon metabolism were measured by liquid chromatography-mass spectrometry. For four time points during the culture, these distributions were used to obtain the best estimates for the metabolic fluxes. The obtained flux fits suggested that the optimally fitted split ratio for the pentose phosphate pathway changed by almost a factor of 2 up and down around a value of 0.27 during the experiment. Statistical analysis revealed that some of the fitted flux distributions for different time points were significantly different from each other, indicating that cell cycle-dependent variations in cytosolic metabolic fluxes indeed occurred.