Methylated DNA Enrichment

Highly sensitive enrichment of methylated DNA
MethylMiner™ Methylated DNA Enrichment Kit enables superior enrichment and differential fractionation of double-stranded DNA based on CpG methylation density, with increased sensitivity over antibody-based methods. Fractionation permits important comparisons between samples and enables researchers to focus analysis on only the methylation densities of interest.

• Partitioning with high affinity binding—at least 4-fold greater sensitivity than antibody-based methods
• Fractionation based on CpG methylation density— ds DNA capture is achieved with MBD2 protein and facilitates ligation of double-stranded adaptors for next-generation sequencing
• Rapid and easy elution with salt eliminates the need for proteinase K treatment and phenol:chloroform extraction
• Precise answers—fractionated DNA permits distinctions to be made regarding methylation status and density
• Fast protocol—completed in less than 4 hours
  
• Easy—simple handling with Dynabeads - the gold standard in magnetic beads

Figure 1— MBD-biotin based CPG methylated DNA affinity Capture. 
The Methyl Miner™ Methylated DNA Enrichment Kit allows for a diversity of elution strategies, depending on you preferred work flow and downstream application. It supports: 1) a single elution using undiluted high salt elution buffer; 2) a single elution using Proteinase K ; 3) a series of step-wise elutions with buffers containing successively greater NaCl concentrations; or 4) step-wise fractional elutions followed by a final Proteinase K treatment. This method overcomes challenges associated with denatured DNA using antibody-based methods. The dsDNA is more compatible with downstream epigenetic analysis applications—including next-generation sequencing.




Figure 2 – MethylMiner™ Methylated DNA Enrichment Kit captures more heavily methylated DNA compared to an antibody-based method. 
Relative capture and recovery of qPCR amplicon 1 DNA by MethylMiner™ Methylated DNA Enrichment Kit and anti-5mC from two different vendors.   In parallel, equal moles of anti-5mC antibody molecules were coupled to M-280 Protein G Dynabeads and MethylMiner MBD-biotin molecules were coupled to M-280 Streptavidin Dynabeads.  After coupling, triplicate aliquots of 10 µL of antibody-beads were incubated with 0.5 µg of fragmented, heat-denatured MCF-7 ssDNA and triplicate aliquots of 10 µL of MethylMiner beads were incubated with 0.5 µg of fragmented, non-denatured dsDNA.  After mixing at 22ºC for one hour, the supernatants were recovered, the beads were washed and then serially eluted with buffers of increasing NaCl concentration.  As a final step, all beads were treated with 20 µg of proteinase K at 56ºC for two hours.  The proteinase K digest supernatants were collected and extracted with phenol:chloroform:isoamyl alcohol (25:24:1).  All fractions were ethanol precipitated, redissolved in water, and assayed with amplicon 1 specific primers by qPCR using a SYBR GreenER mastermix.  Recovery of amplifiable DNA was measured relative to a standard curve of input fragmented genomic DNA.  As shown, ~100% of the  heavily methylated DNA sequence was captured by the MethylMiner beads and ~80% could be eluted with 1 M NaCl.  In contrast, the anti-5mC antibody from two different vendors could only capture ~10-20% of this heavily methylated DNA and elution could only be achieved with proteinase K treatment.