A refined DNA methylation detection method using MspJI coupled quantitative PCR

作者：Petell Christopher J; Loiseau Gilbert; Gandy Ryan; Pradhan Sriharsa; Gowher Humaira^*

来源：Analytical Biochemistry, 2017, 533: 1-9.

DOI：10.1016/j.ab.2017.06.006

• MspJl
• Hpall
• MD-qPCR
• Restriction enzymes
• DNA methylation
• Pluripotency gene enhancers

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 Hpall, respectively. By employing varying concentrations of DNA with methylation ranging from 0 to 100%, our data provide evidence that compared to Hpall, MspJI increases the sensitivity and accuracy of detecting relative DNA methylation gains by MD-qPCR. We also show that the MspJl-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.

• 出版日期2017-9-15
• 单位New England Biolabs