Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation
Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation
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DOI：
10.1038/nprot.2007.271
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Liang, Gangning
中科院分区：
文献类型：
--
作者：
Gonzalgo, Mark L.;Liang, Gangning
甲基化敏感单核苷酸引物延伸（Ms-SNuPE）是一种可用于快速定量单个CpG位点甲基化的技术。用亚硫酸氢钠处理基因组DNA，将未甲基化的胞嘧啶转化为尿嘧啶，同时保持5-甲基胞嘧啶不变。使用Ms-SNuPE进行链特异性PCR以生成用于定量甲基化分析的DNA模板。然后使用寡核苷酸(s)进行SNuPE，这些寡核苷酸被设计成在被询问的CpG位点上游杂交。反应产物在聚丙烯酰胺凝胶上电泳，通过磷酸化图像分析进行可视化和定量分析。Ms-SNuPE技术类似于其他使用亚硫酸氢盐处理基因组DNA以区分未甲基化和甲基化胞嘧啶的定量分析（即COBRA，焦磷酸测序）。Ms-SNuPE可用于高通量甲基化分析和胞嘧啶甲基化的快速定量，适用于广泛的生物学研究，例如检查肿瘤发生过程中的异常甲基化变化，监测DNA甲基化抑制剂诱导的甲基化变化或测量半甲基化。在所需目标序列的PCR扩增和PCR扩增子的制备后，Ms-SNuPE在不到5小时的时间内可以在多达40个样品中询问大约2到4个CpG位点。
Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.