一种用于特定CpG位点甲基化分析的快速、定量、非放射性亚硫酸氢盐-SNuPE-IP RP HPLC检测方法。
A rapid, quantitative, non-radioactive bisulfite-SNuPE- IP RP HPLC assay for methylation analysis at specific CpG sites.
作者信息
El-Maarri Osman, Herbiniaux Ursula, Walter Jörn, Oldenburg Johannes
机构信息
Institute of Experimental Haematology and Transfusion Medicine, Sigmund-Freud Strasse 25, 53105 Bonn, Germany.
出版信息
Nucleic Acids Res. 2002 Mar 15;30(6):e25. doi: 10.1093/nar/30.6.e25.
The precise mapping and quantification of DNA methylation as an epigenetic parameter during development and in diseased tissues is of great importance for functional genomics. Here we describe a rapid, quantitative method to assess methylation levels at specific CpG sites using PCR products of bisulfite-treated genomic DNA. Using single nucleotide primer extension (SNuPE) assays in combination with ion pair reverse phase high performance liquid chromatography (IP RP HPLC) separation techniques, methylated and unmethylated CpGs can be discriminated and quantified based on the different masses and hydrophobicities of the extended primer products. The assay is linear, highly reproducible and several sites can be measured simultaneously in one reaction. It can be semi-automated and eliminates the need for cloning and sequencing of individual bisulfite PCR products.
在发育过程和患病组织中，将DNA甲基化作为一种表观遗传参数进行精确的定位和定量，对于功能基因组学至关重要。在此，我们描述了一种快速、定量的方法，可利用亚硫酸氢盐处理的基因组DNA的PCR产物来评估特定CpG位点的甲基化水平。通过将单核苷酸引物延伸（SNuPE）分析与离子对反相高效液相色谱（IP RP HPLC）分离技术相结合，基于延伸引物产物的不同质量和疏水性，可区分并定量甲基化和未甲基化的CpG。该分析呈线性，具有高度可重复性，并且可以在一个反应中同时测量多个位点。它可以实现半自动操作，无需对单个亚硫酸氢盐PCR产物进行克隆和测序。
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