检测肝细胞核因子1β（HNF1β）基因中三等位基因单核苷酸多态性rs757210的TaqMan检测法的替代方法。
Alternative methods to a TaqMan assay to detect a tri-allelic single nucleotide polymorphism rs757210 in the HNF1β gene.
作者信息
Swen Jesse J, Baak-Pablo Renee F, Guchelaar Henk-Jan, van der Straaten Tahar
机构信息
Department of Clinical Pharmacy and Toxicology, Leiden, University Medical Center, Leiden, The Netherlands.
出版信息
Clin Chem Lab Med. 2011 Oct 21;50(2):279-84. doi: 10.1515/CCLM.2011.758.
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.
背景
多项研究报告称，使用TaqMan探针进行肝细胞核因子1β（HNF1β）rs757210基因分型存在困难。这可能是由于该单核苷酸多态性（SNP）的三等位基因性质所致。本研究的目的是开发rs757210基因分型的替代方法。
方法
开发了焦磷酸测序法和小扩增子高分辨率熔解分析（HRM），并在2型糖尿病患者组（n = 258）和健康献血者组（n = 183）中进行测试。结果通过桑格测序法进行确认。
结果
在2型糖尿病患者组和健康献血者组中，焦磷酸测序法确定的A、G和C等位基因频率分别为0.42、0.56、0.02和0.37、0.62、0.01。使用更常规的HRM方法也得到了类似结果。焦磷酸测序法和HRM的结果一致性为99.6%。
结论
焦磷酸测序法和HRM可用于对HNF1β基因中的三等位基因SNP rs757210进行基因分型，与市售的TaqMan分析相比，它们具有能够确定罕见C等位基因变体的优势。
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