What Are the Advantages of DNA Methylation Detection Kits？

Higher sensitivity

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The MethylMiner' Methylated DNA Enrichment Kit is demonstrably more sensitive than the antibody method, as seen from head-to-head comparison of recovery of natural and synthetic methylated DNA molecules with qPCR assays. In these assays, the MethylMiner' method routinely recovers 4-fold more amplifiable DNA and methylated sequences containing fewer methyl-CpG groups than the antibody method. The MethylMiner' kit can retrieve DNA fragments that contain as few as two methylated CpGs with the same sensitivity that the antibody-based MeDIP (methylated DNA immunoprecipitation) assay yields fragments containing 4 methylated CpGs. 

Enhanced genome-wide methylation 

The greater sensitivity of the MethylMiner' Methylated DNA Enrichment Kit permits recovery of a more complex subset of genomic sequences and, hence, a better representation of the methylation that is present genome-wide within the sample. On CpG island-focused microarrays, the MethylMiner' Kit has yielded 5 times as many high hits (S/N >10) as antibody-based MeDIP. On tiling arrays, the difference is even more pronounced. In all cases tested so far (n=6), high hits that were unique to the MethylMiner' workflow have proven to be true positives as compared to bisulfite-sequencing.

Easy methylome profiling by serial elution
The MethylMiner' Kit can be used to de-convolve the methylome in a manner that is impossible with anti-5-Methylcytosine antibody. This is because MethylMiner'- captured DNA strands can be eluted serially with changes in the salt concentration of the elution buffer.

The kit is scalable depending on how much DNA you would like to use. There are separate protocols in the manual to follow for different input amounts. Our R&D has observed excellent linearity of DNA recovery in the range of 25 ng to 1 µg of input with the recommended micro scale protocol. The smallest amount R&D has tried is 5 ng. Highly methylated sequences can be effectively captured at 5 ng scale, but low- to moderately methylated strands may be difficult to recover at that scale. The kit recommends using 10 µL of beads for every µg of DNA. 200 µL of beads are provided in the kit, so the maximal input would be 20-25 µg DNA. 

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We do not provide specific instructions for how to fragment the DNA, as this depends on specific downstream applications. However, DNA must be fragmented to an average size of less than 1,000 bp and should be in DNase-free water, TE buffer, or another low ionic-strength, neutral pH buffer. Fragmentation of the DNA can be achieved either by complete digestion with the restriction enzyme Mse I, or by physical means such as by sonication. Mse I digestion and sonication with a Bioruptor' sonicator or Covaris' sonicator have been demonstrated to be adequate. Fragmentation with the Covaris' instrument is preferred because it yields reproducible, relatively narrow size distribution of fragments and is not thought to produce fragment-end sequence bias that is expected with enzymatic digestion.

The MBD domain used for our MethylMiner' protein is derived from the human MBD2 protein. The exact sequence is proprietary; however there is a high degree of sequence identity between the MBD domains. Our internal experiments indicate that we have binding affinities that are very close to those reported by Fraga et al (Nucleic Acids Research, ; 31:-). We have cited Fraga et al. as the most persuasive work indicating that the MBD domain from MBD2 protein is possibly the best domain for binding. To our knowledge, there is no study that shows this domain has any sequence biases. 

The MethylMiner' Kit has several advantages over whole-genome bisulfite conversion'based methylome sequencing in terms of template requirements, sample throughout, reagent and instrument-time costs, and scalability:

Less starting template needed
Sequencing with MethylMiner' Kit'enriched DNA requires ten times less DNA mass compared to whole-methylome sequencing after bisulfite treatment. MethylMiner' Kit enrichment typically starts with as little as 1-2 µg of sample genomic DNA and yields 3-20% of the input DNA sample as the 'methyl-CpG enriched' fraction. In contrast, a bisulfite conversion-base workflow would require a lot more starting template: approximately 5-10 µg.

Less sequencing coverage needed
Since the MethylMiner' Kit enrichment yields only a subset of all possible DNA sequences, the amount of sequencing that needs to be conducted to measure these regions at 10-fold coverage is approximately 5-30 fold less than for whole-genome sequencing. In contrast, bisulfite-based methylome sequencing is increased 3-10 fold because the bisulfite treatment is very harsh on the sample, and in order to make an accurate measurement of the degree of methylation at any given cytosine residue, each cytosine position should be independently sequenced more often than without bisulfite conversion.

Reduced cost
The cost per MethylMiner' Kit'processed sample is reduced 5-30 fold as opposed to increased 3-10 fold for bisulfite-based methylome sequencing. If a whole genome costs $6,000 to sequence, a whole-methylome can be expected to cost $18,000-$60,000. However, a MethylMiner' Kit'based profile of the methylation can be obtained for less than $1,200 in sequencing costs.

Higher scalability
The costs for MethylMiner' Kit profiling are also scalable in the sense that a limited amount of sequencing can still yield interpretable results. This is because each sequenced fragment can be interpreted as having contained some degree of CpG methylation. As more sequencing is performed, the overall genome-wide landscape of CpG-methylation becomes more and more defined while the regions having dense methylation become more and more deeply covered.

The HRM calibration is a 3-step procedure. In some instrument software, the calibration process has been streamlined to a single step procedure (applies to the ViiA'7 Real-Time PCR System and QuantStudio' Real-Time PCR Systems). 

For the HRM calibration, the first step is a PCR reaction to generate the HRM dye plate. The reaction plate contains a DNA template, a primer pair, and the PCR mix with HRM dye. The second step is dye calibration using the PCR plate from step 1. The last step is a melt curve run, again using the same plate. For details, please refer to the HRM guides for each instrument:

Want more information on DNA Methylation Detection Kits? Feel free to contact us.

• Fast Real-Time PCR System (with software version 1.4 or 2.0.4 and below)
• Fast Real-Time PCR System (with software version 2.0.5 and above)
• HT Real-Time PCR System
• StepOne' and StepOnePlus' Real-Time PCR Systems
• ViiA' 7 Real-Time PCR System
• QuantStudio' 6 and 7 Real-Time PCR Systems
• QuantStudio' 3 and 5 Real Time PCR Systems
• QuantStudio' 12K Flex Real-Time PCR System