Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex PCR

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 (R² = 0.9997) in buffer diluted samples.