Petell Christopher J, Loiseau Gilbert, Gandy Ryan, Pradhan Sriharsa, Gowher Humaira
Department of Biochemistry, Purdue University, West Lafayette, IN 47907, United States.
College of William and Mary, Williamsburg, VA 23187, United States.
Anal Biochem. 2017 Sep 15;533:1-9. doi: 10.1016/j.ab.2017.06.006. Epub 2017 Jun 15.
DNA methylation is a highly conserved epigenetic modification with critical roles ranging from protection against phage infection in bacteria to the regulation of gene expression in mammals. DNA methylation at specific sequences can be measured by using methylation dependent or sensitive restriction enzymes coupled to semi- or quantitative PCR (MD-qPCR). This study reports a refined MD-qPCR method for detecting gain or loss of DNA methylation at specific sites through the specific use of MspJI or HpaII, respectively. By employing varying concentrations of DNA with methylation ranging from 0 to 100%, our data provide evidence that compared to HpaII, MspJI increases the sensitivity and accuracy of detecting relative DNA methylation gains by MD-qPCR. We also show that the MspJI-coupled MD-qPCR can accurately determine the percent gain in DNA methylation at the Sall4 enhancer and is more sensitive than HpaII in detecting relative gains in DNA methylation at the Oct4 proximal enhancer during embryonic stem cell (ESC) differentiation. The high specificity and sensitivity of this targeted approach increases its potential as a diagnostic tool to detect relatively smaller gains in DNA methylation at specific sites from limited amounts of sample.
DNA甲基化是一种高度保守的表观遗传修饰，其作用至关重要，从细菌中抵御噬菌体感染到哺乳动物中基因表达的调控。特定序列的DNA甲基化可以通过使用依赖甲基化或敏感的限制性内切酶与半定量或定量PCR（MD-qPCR）相结合来测量。本研究报告了一种改进的MD-qPCR方法，分别通过特异性使用MspJI或HpaII来检测特定位点DNA甲基化的增加或减少。通过使用甲基化程度从0到100%的不同浓度DNA，我们的数据表明，与HpaII相比，MspJI提高了MD-qPCR检测相对DNA甲基化增加的灵敏度和准确性。我们还表明，与MspJI偶联的MD-qPCR可以准确测定Sall4增强子处DNA甲基化的增加百分比，并且在检测胚胎干细胞（ESC）分化过程中Oct4近端增强子处DNA甲基化的相对增加方面比HpaII更敏感。这种靶向方法的高特异性和灵敏度增加了其作为诊断工具的潜力，可从有限量的样本中检测特定位点相对较小的DNA甲基化增加。
