DNA Methylation Protocol
DNA methylation status detection is one of the important technique in epigenetics study. Gene promoter region hypermethylation is very common and is associated with inactivation of affected gene. The 5’ gene promoter region contains CpG islands which is the target of DNA methyltransferase. The enzyme methylates cytosine to 5-methylcytosine to regulate the gene expression. DNA methylation is important in both normal embryonic development and in abnormal cancer development. DNA methylation detection methods can be divided into methylation-sensitive restriction endonucleases method and bisulfate modification of cytosine method.
Principle of methylation-sensitive restriction method is that the mehtylation-sensitive restriction enzymes can not cut the methylated DNA site. The bisulfate modification method utilizes the fact that methylated cytosine can not be modified by bisulfate, whereas unmethylated cytosine will be changed to uracile.
DNA methylation detection protocol
This protocol is based on methylation-sensitive restriction endonuclease method.
- Extract sample DNA according to general protocol (5 ug gDNA)
- Decide the final digestion volume and setup the restriction endonuclease digestion of sample DNA.
- 5 ug sample DNA, 1/10 volume of reaction buffer, 1/10 volume of BSA, mix
- Add sterile distilled H2O to the final volume
- Add 2ul of restriction enzyme (10 U/ul).
- Incubate overnight at 37C.
- Run gel electrophoresis to confirm the digestion.
- Add 2 ul of methylation sensitive Eco52I (5 U/ul)
- Incubate 12 h at 37C. Add 2 ul of Eco52I and continue incubation at 37C for 12 h.
- Ethanol precipitation followed by resuspension of digested DNA in 16 ul TE buffer.
- Prepare 0.7% agarose gel (TAE) in the gel apparatus.
- Mix digestion with 4 ul of 5x loading buffer. Run gel electrophoresis in 1X TAE buffer. Confirm sufficient digestion on UV transilluminator.
- Southern blotting and hybridization using the appropriate probe.
- X-film development and result interpretation: The double digestion of DNA with restriction enzyme and the methylation sensitive Eco52I will generate different DNA fragments in methylated DNA sample as compared to unmethylated DNA. This can be detected by appropriate DNA probe.
- Note: appropriate controls should be included：
- Control unmethylated DNA digested with RE alone
- Control unmethylated DNA digested with both RE and Eco52I.
- Molecular Biology