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

Kerékgyártó, Márta and Németh, Nóra and Kerekes, Tamás and Rónai, Zsolt and Guttman, András (2013) Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis. Journal of chromatography. A, 1286. pp. 229-234. ISSN 1873-3778

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Abstract

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.

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