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MethylMagnet® Me-CpG DNA Isolation Kit

Cat# MM101-K12    12 reactions

Cat# MM101-K30    30 reactions

MethylMagnet User Manual

For pricing and to place an order, please contact info@ribomed.com.

The MethylMagnet Me-CpG DNA Isolation Kit is used to isolate methylated DNA from a genomic sample. The MethylMagnet kit utilizes glutathione magnetic beads and can be used to isolate Me-CpG DNA from 1 ng or less of genomic DNA, or it can be scaled up to isolate much larger quantities. After capture, methylated DNA can be eluted several ways. These include:

A. Elution of the intact GST-MBD-mCpG DNA complex with Glutathione

B. Elution of free DNA and denatured GST-MBD with heat

C. Elution of free DNA and digested GST-MBD with Proteinase K

D. Elution of the MBD-mCpG DNA complex and GST with Thrombin

E. Elution of  just the mCpG DNA with salt

High Sensitivity

MethylMagnet can be used even with small DNA sample sizes. Shown here, HeLa genomic DNA and artificially methylated HeLa DNA were incubated with GST-MBD magnetic beads for 1 hour. Bound DNA was eluted from the beads with glutathione after removal of the supernatant.  Eluted DNA samples were tested for the presence of p16 DNA by PCR and analyzed by gel electrophoresis and ethidium bromide staining. The lowest DNA amount (2 ng) which could be visualized by staining corresponds to approximately 300 cells. Even less DNA can be isolated and detected using abscription and RiboMed’s MethylMeter Kits.

 

How It Works

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

The methylated DNA can be eluted from the magnetic beads several ways.

A. Elution of the intact GST-MBD-mCpG DNA complex with Glutathione

B. Elution of free DNA and denatured GST-MBD with heat

C. Elution of free DNA and digested GST-MBD with Proteinase K

D. Elution of the MBD-mCpG DNA complex and GST with Thrombin

E. Elution of  just the mCpG DNA with salt

A.  Elution of the GST-MBD-mCpG DNA complex with Glutathione (PCR or bisulfite ready)

Addition of glutathione displaces the GST-MBD-mCpG DNA complex from the magnetic beads without disrupting the interaction between the DNA and the GST-MBD complex. This DNA can be diluted directly into PCR reactions, during which the GST-MBD protein is heat denatured and dissociates from the DNA. The DNA can also be treated with bisulfite for amplification and bisulfite sequencing. However, because the protein-DNA complex is maintained, the GST tag can then be used for further analysis. The GST can be visualized with a number of different types of labeled GST antibodies, as in our Global Methylation Assay.

B.  Elution of free DNA and denatured GST-MBD with heat (PCR or bisulfite ready)

Captured DNA can be eluted from the beads by heating the samples. Elution by this method releases the double-stranded mCpG DNA and the intact GST-MBD protein in a heat-denatured form. Samples are ready for PCR or other analysis without further treatment.

C.  Elution of free DNA and digested GST-MBD with Proteinase K

The methylated DNA can be released from the glutathione beads by digesting the GST-MBD protein through which it is attached. This results in the release of the DNA and amino acids and peptides. The Proteinase K must then be inactivated before proceeding with most downstream applications. This is accomplished by heat inactivation or the addition of a Proteinase K inhibitor.

D. Elution of the MBD-mCpG DNA complex with Thrombin

 The location of a thrombin cleavage site between the GST and MBD domains allows the DNA to be released as a complex with the MBD. Thrombinis a serine protease that catalyzes many blood coagulation reactions.  It has high specificity for the consensus sequence Leu-Val-Pro-Arg-Gly-Ser, cleaving the peptide bond between Arg and Gly. Because this site is found infrequently in proteins, it is used in fusion proteins to allow release of the overexpressed protein from the tag, in this case GST. Two options are available for retrieval of methylated DNA with thrombin. The first involves elution of the protein from the column by including thrombin in the elution buffer. Because of the infrequency of the thrombin cleavage site in other proteins, for most applications, it is not necessary to inactivate or remove the thrombin. Should it be necessary, however, biotin-tagged thrombin is also available, which can easily be removed from the column elution fraction with streptavidin magnetic beads. An alternative method involves releasing the GST-MBD-DNA complex from the column with glutathione and then using immobilized thrombin, also commercially available, to release the GST. This allows for easy separation of the MBD-DNA from the thrombin.

E. Elution of  mCpG DNA with salt

The methylated DNA can be released from the MBD and eluted from the column with salt. All of the DNA can be eluted with using high salt. Alternatively, the DNA can be stepped off the column with elution buffers containing different salt concentrations, resulting in fractionation of the mCpG by the level of island methylation. This method does have the disadvantage of diluting the DNA and may require precipitation before downstream applications.