Can I purchase the Dynabeads M-280 Streptavidin from the MethylMiner Methylated DNA Enrichment Kit as a standalone item?
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).
The MethylMiner Methylated DNA Enrichment Kit (Cat. No. ME10025) cannot directly give single nucleotide resolution of the methylation pattern, so why should I use it?
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.
With 200 ng DNA input, I am getting non-methylated DNA carryover. Why is this?
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.
What program can I use to design primers for methylation mapping experiments?
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/).
Why should I use the MethylMiner Methylated DNA Enrichment Kit instead of utilizing whole-genome bisulfite conversion and methylome sequencing?
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.
