BACKGROUND: Several studies report difficulties in genotyping HNF1β rs757210 using TaqMan probes. This is possibly due to the tri-allelic nature of this single nucleotide polymorphism (SNP). The aim of the present research was to develop alternative methods for genotyping rs757210. METHODS: Pyrosequencing and high resolution melting analysis of small amplicons (HRM) were developed and tested in panels of type 2 diabetes mellitus patients (n=258) and healthy blood donors (n=183). Results were confirmed by Sanger sequencing. RESULTS: With pyrosequencing, allele frequencies for the A, G and C allele of 0.42, 0.56, 0.02 and 0.37, 0.62, 0.01 were established in the panel of type 2 diabetes mellitus patients and healthy blood donors, respectively. Similar results were found using the more routinely available HRM method. Results for pyrosequencing and HRM were in 99.6% concordance. CONCLUSIONS: Pyrosequencing and HRM can be used to genotype the tri-allelic SNP rs757210 in the HNF1β gene and have the advantage over the commercially available TaqMan analysis that they can determine the rare C-allele variant.
BACKGROUND: Several studies report difficulties in genotyping HNF1β rs757210 using TaqMan probes. This is possibly due to the tri-allelic nature of this single nucleotide polymorphism (SNP). The aim of the present research was to develop alternative methods for genotyping rs757210. METHODS: Pyrosequencing and high resolution melting analysis of small amplicons (HRM) were developed and tested in panels of type 2 diabetes mellituspatients (n=258) and healthy blood donors (n=183). Results were confirmed by Sanger sequencing. RESULTS: With pyrosequencing, allele frequencies for the A, G and C allele of 0.42, 0.56, 0.02 and 0.37, 0.62, 0.01 were established in the panel of type 2 diabetes mellituspatients and healthy blood donors, respectively. Similar results were found using the more routinely available HRM method. Results for pyrosequencing and HRM were in 99.6% concordance. CONCLUSIONS: Pyrosequencing and HRM can be used to genotype the tri-allelic SNP rs757210 in the HNF1β gene and have the advantage over the commercially available TaqMan analysis that they can determine the rare C-allele variant.
Authors: Judy M Obliosca; Sara Y Cheng; Yu-An Chen; Mariana F Llanos; Yen-Liang Liu; Darren M Imphean; David R Bell; Jeffrey T Petty; Pengyu Ren; Hsin-Chih Yeh Journal: J Am Chem Soc Date: 2017-05-09 Impact factor: 15.419
Authors: Krishna K Gopaul; Jessica Sells; Robin Lee; Stephen M Beckstrom-Sternberg; Jeffrey T Foster; Adrian M Whatmore Journal: BMC Res Notes Date: 2014-12-11