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View additional product information for MethylMiner™ Methylated DNA Enrichment Kit - FAQs (ME10025)
11 product FAQs found
Yes, the Dynabeads M-280 Streptavidin from the MethylMiner Methylated DNA Enrichment Kit is available as a standalone item and the Cat. Nos. are 11205D (2 mL), 11206D (10 mL), and 60210 (100 mL).
In many studies, the immediate goal is to identify, genome-wide methylation differences between samples. For such studies, enrichment of methylated sequences can be an enabling technology that brings the desired data within economic reach.
Minimal Bias:
The MethylMiner Kit is a sensitive enrichment technology that samples the CpG methylation in a minimally biased manner-thus it is an excellent discovery and characterization tool. It is readily compatible with next-generation sequencing workflows, as well as with microarray and PCR-based genome surveying technologies.
Compatible with next-generation sequencing:
The resolution of methylation detection by the MethylMiner Kit is a function of the fragmentation of the DNA sample prior to enrichment, but can be practically stated as ~100 bp. This makes it uniquely well-matched with the short-read, high-throughput SOLiD System sequencing technology. In many cases, this degree of resolution may be sufficient to identify variation if genome methylation is biologically significant.
Towards single nucleotide resolution:
Once differences and similarities between the methylation patterns of the samples of interest have been identified at the 100 bp resolution, it becomes much more practical to design and utilize cost-effective targeted re-sequencing strategies to determine the methylation patterns at single nucleotide resolution. Enrichment is absolutely required in order to screen the hundreds to thousands of samples that must be surveyed in the study of the complex human diseases that are the targets of DNA methylation research. In contrast, using antibody methods require and significant quantities of DNA, significant amounts of sequencing. Much of the methylation pattern is relatively invariant between samples and thus much of this investment is likely to be wasted on generating redundant information about constitutively methylated sequences.
When there is very little or no methylated DNA, MBD protein also binds non-methylated DNA to some extent. So it is important to follow the right protocol when using low DNA input. The product manual (https://tools.thermofisher.com/content/sfs/manuals/methylminer_man.pdf) has different protocols specified for different DNA input amounts.
You can use Methyl Primer Express Software to design primers for methylation studies. You can download the program for free from here (http://resource.thermofisher.com/page/WE28396_1/).
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 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, 2003; 31:1765-1774). 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.
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.
Typical total yields of mammalian CpG-methylated DNA are 3-20% of the input mass of DNA, or 0.3-5.0 µg when starting with 10-25 µg. Yield is depends on the method of DNA fragmentation. Fragmenting to relatively short lengths will reduce the total mass recovered. Also, if any of the DNA is denatured or reduced to tiny oligos and nucleotides by the fragmentation, this "degraded" DNA will not bind the MethylMiner beads efficiently, and thus the yield will be lower.
Most commercial available gDNA isolation products can be used and the PureLink Genomic DNA Mini Kit works well. Make sure to work in DNase-free conditions, and that the gDNA remains double-stranded. The MBD2 protein will not bind to ssDNA.
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.
Higher sensitivity
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.