Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE)
Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE)
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DOI：
10.1093/nar/25.12.2529
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发表时间：
1997-06-15
影响因子：
14.9
通讯作者：
Jones, PA
中科院分区：
文献类型：
--
作者：
Gonzalgo, ML;Jones, PA
我们开发了一种快速定量方法（Ms-SNuPE），用于评估特定CpG位点的甲基化差异，该方法基于亚硫酸氢盐处理DNA，然后进行单核苷酸引物延伸。基因组DNA首先与亚硫酸氢钠反应，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。然后使用对亚硫酸氢盐转化的DNA特异性的PCR引物进行所需靶序列的扩增，分离所得产物并用作模板用于在感兴趣的CpG位点处进行甲基化分析。这种甲基化敏感的技术与现有的用于检测甲基化变化的方法相比具有几个优点，因为可以分析少量的DNA，包括显微解剖的病理切片，并且它避免了利用限制性内切酶来确定CpG位点的甲基化状态。
We have developed a rapid quantitative method (Ms-SNuPE) for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension. Genomic DNA was first reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence was then performed using PCR primers specific for bisulfite-converted DMA and the resulting product isolated and used as a template for methylation analysis at the CpG site(s) of interest. This methylation-sensitive technique has several advantages over existing methods used for detection of methylation changes because small amounts of DNA can be analyzed including microdissected pathology sections and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.