CSIRO Food and Nutritional Sciences, Preventative Health Flagship, North Ryde, NSW, Australia.
Biotechniques. 2010 Oct;49(4):xiii-xvii. doi: 10.2144/000113494.
We describe a new method that is well-suited for the determination of the methylation level of repetitive sequences such as human Alu elements. We have applied the method to the analysis of cell and tissue DNAs and expect it to have wide utility in studies of DNA methylation in cancer and other disease states, in monitoring response to epigenetic cancer therapies and in epidemiological studies. Only 1 ng DNA is needed for a duplex, one-tube real-time PCR in which methylation level and the amount of input DNA are concurrently measured. The relative cutting by the methylation-sensitive enzyme BstUI is compared with that of the methylation-insensitive enzyme DraI to give a measure of DNA methylation. The method depends upon the use of 5'-tailed, 3'-blocked oligonucleotides called facilitator oligonucleotides (Foligos). Only cut DNAs with specific matching sequences at their 3' ends can copy the tails of the Foligos and thus become tagged and available for subsequent PCR. Both the tagging and PCR are carried out by the same enzyme, Taq DNA polymerase. Because amplification only occurs if suitable ends have been generated in the target DNA, we have called this method end-specific PCR (ESPCR). ESPCR avoids the bisulfite treatment step that is usually required to measure methylation.
我们描述了一种新方法，该方法特别适用于重复序列（如人类 Alu 元件）的甲基化水平的测定。我们已经将该方法应用于细胞和组织 DNA 的分析，预计它将在癌症和其他疾病状态下的 DNA 甲基化研究、监测针对表观遗传癌症治疗的反应以及流行病学研究中具有广泛的应用。在双管实时 PCR 中，仅需 1ng DNA 即可进行单管反应，同时测量甲基化水平和输入 DNA 的量。通过比较甲基化敏感酶 BstUI 的切割与甲基化不敏感酶 DraI 的切割，得出 DNA 甲基化的衡量标准。该方法依赖于使用 5'-端加尾、3'-端封闭的寡核苷酸，称为促进寡核苷酸（Foligos）。只有在其 3'末端具有特定匹配序列的切割 DNA 才能复制 Foligos 的尾部，从而被标记并可用于随后的 PCR。标记和 PCR 都是由相同的酶 Taq DNA 聚合酶完成的。由于扩增仅在目标 DNA 中生成合适的末端时才会发生，因此我们将这种方法称为末端特异性 PCR（ESPCR）。ESPCR 避免了通常用于测量甲基化的亚硫酸氢盐处理步骤。
