The MethylMagnet mCpG DNA Isolation Kit is used to isolate methylated DNA from a genomic sample. The MethylMagnet kit utilizes the MethylMagnet GST-MBD fusion protein and glutathione magnetic beads for capture of methylated DNA. It can be used to isolate mCpG DNA from 1 ng to 1 µg of genomic DNA, or it can be scaled up to isolate much larger quantities. After capture, methylated DNA can be eluted several ways.
Step 1: DNA Fragmentation
DNA must first be fragmented so that the region of interest is physically separated from other regions on DNA that may be methylated. The island detection assays that utilize abscription for detection using the MethylMeter kits have been designed to work with DNA that has been cut with the restriction endonuclease Mse I. Mse I cuts at the sequence TTAA and therefore is not affected by CpG methylation. However, MethylMagnet can be used with DNA that has been fragmented by other methods or by restriction with other enzymes.
Step 2: Capture of Methylated DNA
The fragmented DNA is first incubated with the MethylMagnet GST-MBD protein which has already been attached to glutathione magnetic beads. The DNA population will generally contain a mixture of CpG islands which are methylated and those that are not. Those which are methylated will bind preferentially to the beads via interaction of the mCpG sites and the MBD domain. Incubation is for 1 hour at room temperature. After binding, the column is washed to remove any non-specifically bound DNA.
Step 3: Elution of Methylated DNA
- Elution of the GST-MBD-mCpG DNA complex with Glutathione
- Elution of free DNA and denatured GST-MBD with heat
- Elution of free DNA and digested GST-MBD with Proteinase K
- Elution of the MBD-mCpG DNA complex and GST with Thrombin
- Elution of just the mCpG DNA with salt