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首页> 外文期刊>Human mutation >Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension-application to the variants of mannose-binding lectin gene.
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Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension-application to the variants of mannose-binding lectin gene.

机译：光蛋白水母发光蛋白作为通过引物延伸应用于甘露糖结合凝集素基因变异的SNP基因分型的新型报告基因。

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摘要

Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.-619G>C and c.-290G>C (promoter region), and one SNP at position c.-66C>T of the 5' untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5' end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3 sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data.
机译：甘露糖结合凝集素（MBL）是先天免疫系统的关键组成部分，其缺乏与对各种感染和自身免疫性疾病的敏感性增加有关。由于甘露糖结合凝集素2基因（MBL2）中的几个核苷酸变异与MBL的功能缺陷有关，因此越来越需要筛选其等位基因变体并开发MBL2的基因分型方法。在这种情况下，我们提出了一种快速，鲁棒，经济高效且可自动化的方法，用于检测MBL2的所有已知等位基因变体。本报告首次引入了光蛋白水母发光蛋白作为引物延伸（PEXT）反应进行基因分型的报告基因。该方法涉及跨越所有六个变异核苷酸位点的基因组区域的单次PCR扩增，即外显子1中的三个结构突变（c.154C> T，pArg52Cys; c.161A> G，p.Gly54Asp和c.170A） > G，p.Gly57Glu），在c.-619G> C和c.-290G> C（启动子区域）位置有两个单核苷酸多态性（SNP），在5'的c.-66C> T位置有一个SNP。非翻译区域。对于每个位点，PCR之后都进行PEXT反应。生物素-dUTP被掺入扩展引物中。基因分型引物在其5'末端包含一个poly（dA）片段。通过在包被有聚（dT）-白蛋白的微量滴定孔的表面上杂交来捕获产物。仅通过与链霉亲和素-水母发光蛋白缀合物反应来检测延伸的引物。只需添加Ca2 +，即可在3秒内测量结合的光蛋白水母发光蛋白。我们对PEXT反应进行了广泛的优化研究，并使用来自27个正常DNA样本的血液样本对六个核苷酸变异位点进行了基因分型。该方法的结果与测序数据完全吻合。

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