title: Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis.
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.
authors.affiliation: Horváth Laboratory of Bioseparation Sciences, University of Debrecen, H-4032 Debrecen, Nagyerdei krt. 98, Hungary.
authors.forename: Márta
authors.initials: M
authors.lastname: Kerékgyártó
authors.forename: Nóra
authors.initials: N
authors.lastname: Németh
authors.forename: Tamás
authors.initials: T
authors.lastname: Kerekes
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Rónai
authors.forename: András
authors.initials: A
authors.lastname: Guttman
mesh_terms: D001665:Binding Sites; D019075:Electrophoresis, Capillary / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods*; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008565:Membrane Proteins / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results
chemical_list: D008565:Membrane Proteins; D035683:MicroRNAs; C114987:wolframin protein