基于微阵列的功能单核苷酸多态性基因分型方法，采用双色荧光杂交技术。

Microarray-based method for genotyping of functional single nucleotide polymorphisms using dual-color fluorescence hybridization.

作者信息

Ji Meiju, Hou Peng, Li Song, He Nongyue, Lu Zuhong

机构信息

Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.

出版信息

Mutat Res. 2004 Apr 14;548(1-2):97-105. doi: 10.1016/j.mrfmmm.2004.01.002.

DOI:10.1016/j.mrfmmm.2004.01.002

PMID:15063140

Abstract

Screening disease-related single nucleotide polymorphism (SNP) markers in the whole genome has great potential in complex disease genetics and pharmacogenetics researches. It has led to a requirement for high-throughput genotyping platforms that can maximize the efficient screening functional SNPs with respect to accuracy, speed and cost. In this study, we attempted to develop a microarray-based method for scoring a number of genomic DNA in parallel for one or more molecular markers on a glass slide. Two SNP markers localized to the methylenetetrahydrofolate reductase gene (MTHFR) were selected as the investigated targets. Amplified PCR products from nine genomic DNA specimens were spotted and immobilized onto a poly-l-lysine coated glass slide to fabricate a microarray, then interrogated by hybridization with dual-color probes to determine the SNP genotype of each sample. The results indicated that the microarray-based method could determine the genotype of 677 and 1298 MTHFR polymorphisms. Sequencing was performed to validate these results. Our experiments successfully demonstrate that PCR products subjected to dual-color hybridization on a microarray could be applied as a useful and a high-throughput tool to analyze molecular markers.

摘要

在全基因组中筛选疾病相关单核苷酸多态性（SNP）标记物在复杂疾病遗传学和药物遗传学研究中具有巨大潜力。这就需要高通量基因分型平台，该平台能够在准确性、速度和成本方面最大限度地高效筛选功能性SNP。在本研究中，我们尝试开发一种基于微阵列的方法，用于在载玻片上同时对多个基因组DNA进行一个或多个分子标记物的评分。选择定位于亚甲基四氢叶酸还原酶基因（MTHFR）的两个SNP标记物作为研究对象。将来自九个基因组DNA样本的扩增PCR产物点样并固定在聚-L-赖氨酸包被的载玻片上以制备微阵列，然后通过与双色探针杂交进行检测以确定每个样本的SNP基因型。结果表明，基于微阵列的方法能够确定MTHFR基因677和1298位点多态性的基因型。进行测序以验证这些结果。我们的实验成功证明，在微阵列上进行双色杂交的PCR产物可作为一种有用的高通量工具来分析分子标记物。