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.
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.
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