Light-emitting diode induced fluorescence (LED-IF) detection design for a pen-shaped cartridge based single capillary electrophoresis system.

CGE is a well-established separation technique for the analysis of biologically important molecules such as nucleic acids. The inherent high resolving power, rapid analysis times, excellent detection sensitivity, and quantification capabilities makes this method favorable compared to conventional manual polyacrylamide and agarose slab gel electrophoresis techniques. In this paper we introduce a novel single-channel capillary gel electrophoresis system with LED-induced fluorescence detection also utilizing a compact pen-shaped capillary cartridge design for automatic analysis of samples from a 96-well plate. To evaluate the suitability of the system, 1000 genomic DNA(gDNA) samples were analyzed in gel filled capillaries and detected by the microball ended excitation and emission optical fiber based LED-induced fluorescence detection system. Excellent migration time reproducibility of RSD <0.75% was obtained over the course of 1000 runs. The system rapidly distinguished between intact and degraded gDNA samples, therefore provided important information if they could be used for downstream quantitative PCR processing where high-quality intact gDNA was key. We envision that this novel system design will rapidly find new applications in both research and clinical diagnostic laboratories as a highly sensitive and easy to use bio-analytical approach.

Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis.

The transmembrane protein wolframin (WSF1) plays a crucial role in cell integrity in pancreatic beta cells and maintaining ER homeostasis. Genetic variations in the WFS1 gene have been described to be associated with Wolfram syndrome or type 2 diabetes mellitus. In this paper we report on an efficient double-tube allele-specific amplification method in conjunction with ultrafast capillary gel electrophoresis for direct haplotyping analysis of the SNPs in two important miRNA-binding sites (rs1046322 and rs9457) in the WFS1 gene. An automated single-channel capillary gel electrophoresis system was utilized in the method that provided dsDNA fragment analysis in less than 240 s. The light-emitting diode induced fluorescence (LEDIF) detection system enabled excellent sensitivity for automated haplotyping of a large number of clinical samples. The detection limit was 0.002 ng/µL using field amplified injection from water diluted samples. The dynamic quantitation range was 0.08-10.00 ng/µL (R(2)=0.9997) in buffer diluted samples.