DNA Methylation Analysis Support—Getting Started

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.

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. 

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.

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.

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, 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. 

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.

When using the MethylCode™ Bisulfite Conversion Kit, 500 pg-2 μg of genomic DNA is needed. For optimal conditions, use 200-500 ng. Please note that for GC-rich regions, sample amounts greater than 500 ng may result in incomplete bisulfite conversion.

Starting DNA amounts from 50 pg up to 2 μg can be used for DNA conversion using the EpiJET Bisulfite Conversion Kit. Nevertheless, for optimal results, use 200–500 ng of input DNA. High input DNA amounts may result in incomplete bisulfite conversion for some GC-rich regions. 

This is usually not very successful. In general, crosslinked or damaged DNA is poor starting material.

No, residual RNA does not interfere with the bisulfite conversion reaction.

Yes, bisulfite conversion requires DNA denaturation. Supercoiled DNA (usually of plasmid origin) is typically more difficult to denature, which may lead to potential under-conversion.

The CT Conversion Reagent that has been prepared in solution may be stored up to one week at -20°C. We recommend thawing at room temparature and mixing for 2 mins by rotating or vortexing before use. 

Yes. There is no upper limit to the volume that can used to elute. If there is insufficient elution buffer remaining, then water or TE can be used to elute.

The converted DNA is stable for one day at room temperature, one week at 4°C, and two to four months at –20°C. We recommend storing your converted DNA below –70°C whenever possible.
Note: The DNA is single-stranded and inherently less stable than dsDNA.

The best conversion results are obtained with highly pure DNA. Most commercially available kits yielding good quality DNA can be used. The PureLink™ Genomic DNA Purification Kit is a complete kit we offer for the isolation of genomic DNA.

• Protocol A results in DNA conversion efficiency >99%, is more sensitive and gives lower DNA degradation levels than Protocol B. This protocol is recommended for all routine epigenetic applications. 
• Protocol B results in DNA conversion efficiency >95% and is much faster than Protocol A (30 min vs. 160 min for DNA denaturation/bisulfite conversion step). Modified DNA is suitable for PCR of amplicons up to 300 bp. This protocol is not recommended for small DNA amounts (less than 50 ng of input DNA). It is the best for fast DNA methylation screening. 

The powdered chemicals will add to the total volume of the final solution. The volume should be greater than 1.4 mL after completely resuspending the CT Conversion Reagent as indicated in the protocol.

Yield of DNA after bisulfite conversion (when input of DNA is 200-500 ng) is ~70-80%. We suggest performing qPCR or agarose gel electrophoresis and comparing your sample to a known amount of DNA. Since the bisulfite converted DNA strands are no longer complementary, this DNA mainly stays in a single stranded form and resembles RNA. So if spectrophotometer method is used, converted DNA concentration needs to be calculated using 40 μg/mL for absorbance at 260 nm = 1.0. 

Yes. We have tried using the EpiJET Bisulfite Conversion Kit with next-generation sequencing, and it works really well (conversion of unmethylated Cs was >99.98% when using Protocol A; the 30 min protocol is not recommended for next-generation sequencing). For next-generation sequencing support, visit our Next-Generation Sequencing Support Center.

Yes. For more information, please go here.

Bisulfite modification is harsh and may cause strand breaks. Most research publications recommend 200 base pair lengths; however, larger fragments have been amplified with an optimized protocol.

We have found that 150 μL works fine for the conversion reaction with most PCR machines.

Among the Applied Biosystems™ real-time PCR instruments, the following are compatible with HRM: 

• 7500 Fast Real-Time PCR System
• 7900HT Fast 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™ 12K Flex Real-Time PCR System
• QuantStudio™ 3 and 5 Real-Time PCR Systems

The instrument will need to be calibrated for HRM. This includes a HRM dye calibration and a melt curve file for data analysis. You can find the appropriate HRM calibration plate for your instrument here.

We offer the MeltDoctor™ HRM dye for HRM application. Other commonly used dyes for HRM include LCGreen™ Plus, Eva Green™ dye and SYBR™ Green dye. 

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:

• 7500 Fast Real-Time PCR System (with software version 1.4 or 2.0.4 and below)
• 7500 Fast Real-Time PCR System (with software version 2.0.5 and above)
• 7900HT 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

You can use Methyl Primer Express™ Software to design primers for methylation studies. You can download the program for free from here.

The PCR primers need to be designed for robust performance and must be specific to the region of interest with a minimum of primer-dimers and non-specific products. A variety of software tools are available to help design HRM primers, including Primer Express™ v3.0 Software, Primer3, Primer3Plus, and Methyl Primer Express™ Software for methylation-sensitive HRM analysis.

No. We do not provide a primer design service for HRM. 

We provide two options for HRM reagents:

• The MeltDoctor™ HRM Master Mix (Cat. No. 4415440) contains all components (excluding template and primers) formulated for superior HRM performance across a wide range of genomic targets. It is recommended for most HRM applications except methylation-sensitive HRM analysis. 
• The MeltDoctor™ HRM Reagent Kit (Cat. No. 4425557) provides individually packaged PCR components and the MeltDoctor™ HRM Dye required for HRM. It is recommended for methylation-sensitive HRM analysis (as it does not contain dUTP). 

Yes, if you are using HRM for genotyping, then you can use our MeltDoctor™ HRM Positive Control Kit (Cat. No. 4410126) to help troubleshoot your experiment. The kit contains control gDNA for each allele (A, A/G, and G), along with a HRM Primer Mix to match.

We do not have any HRM calibration plates for 96-well standard blocks. To perform HRM experiments on such a system, you can instead use our MeltDoctor™ HRM Calibration Standard (Cat. No. 4425562) to create your own calibration plate. 

The Thermo Fisher Cloud HRM App is compatible with *.eds files from the following instruments:

• 7500 and 7500 Fast Real-Time PCR Systems
• 7900HT Fast 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™ 12K Flex Real-Time PCR System
• QuantStudio™ 3 and 5 Real-Time PCR Systems

Among the Applied Biosystems™ real-time PCR instruments, the following are compatible with HRM: 

• 7500 Fast Real-Time PCR System
• 7900HT Fast 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™ 12K Flex Real-Time PCR System
• QuantStudio™ 3 and 5 Real-Time PCR Systems