Since their debut with tiling microarrays back in 2005, DNA methylation affinity reagents have rapidly become a weapon of choice for prepping samples for genome-wide methylation studies. In the last few years enrichment methods have evolved almost as rapidly as the technology platforms on which they’re analyzed. Let’s take a look at some of the recent developments.
Affinity Abbreviated: MeCP2, MeDIP, and MCIp
Adrian Bird’s lab got the DNA methylation enrichment ball rolling back in 1994 by pairing up MeCP2 and affinity chromatography to separate methylated from unmethylated DNA fragments. The procedure took a lot of time and genomic DNA, two ingredients that are in short supply today, but like all good predecessors it laid the foundation for the more recent approaches.
Two offspring that have sprung up more recently are Methylated DNA Immunoprecipitation (MeDIP) and Methyl CpG Immunoprecipitation (MCIp). Besides having catchy aliases, they both use affinity reagents that stick to methylated DNA like Velcro. MeDIP uses a 5’MeC antibody while MCIp snags methylated DNA with the methyl-CpG binding domain (MBD) of the MBD2 methyl binding protein.
The MeDIP approach was similar to ChIP protocols that had been a staple in the chromatin community for years, so it wasn’t that foreign to researchers. It really streamlined genome-wide methylation analysis as it didn’t require knowing which restriction enzymes were going to cut where and reduced the headaches from trying to optimize the specificity of microarray probes for bisulfite converted DNA (3-bases). As a result, the MeDIP approach was embraced early and made its way into a number of lab protocols and commercial microarray provider’s manuals.
Move Over MeDIP?
While MeDIP has had a head start within the research community, we’re betting* that MBD-based approaches will turn up in a lot more labs in the near future. Here’s why:
- Reason 1: MBD binds double stranded DNA so you can pull down your regions of interest without worrying about denaturing conditions or running into issues quantifying your material afterwards. Cool Bonus: This also means that the enriched material is ready to head into high throughput sequencing (HTS) libraries with minimal additional fiddling. The ssDNA that comes off of MeDIP reactions requires more manipulation before it’s ready for sequencing.
- Reason 2: MBD Elution Process is more flexible than a Cirque de Soleil performer and doesn’t take years to master. Depending on your downstream application, you can opt for a high salt elution instead of the overnight Proteinase K treatment in MeDIP protocols if you’re pressed for time, or fractionate your regions of interest based on their degree of methylation. Fractionation might be reason enough for many researcher to give this approach a test-drive which leads us to…
- Reason 3: DNA can be separated by methylation density by eluting with a varying salt gradient. Compared to the all-or-none elution of the antibody approach, fractionation lets you investigate regions that have intermediate methylation densities, and study subtle differences in samples.
The Changing Methylation Landscape
Recent studies are indicating that differential and tissue specific methylation patterns frequent low to medium density CpG islands (Rehli et al, 2009, Weber et al 2007). Moreover, the regions where the interesting methylation action is can be outside CpG islands and in some cases, a long hike from promoters (Irizarry et al 2009 and Rehli et al, 2009).
Researchers who want the full picture of genome-wide DNA methylation (CpG islands, CpG shores, gene body methylation, distal enhancers etc.,), will definitely want to consider the sequencing route for the coverage and an MBD based method to pull down and fractionate some of the interesting regions of variable CpG density and methylation. MBD enrichment is well poised for this challenge.
Dr. Michael Rehli, whose team recently identified interesting methylation patterns where you wouldn’t necessarily expect, shared his thoughts on using MBDs with us recently. “People using MeDIP, I think, are pretty frustrated because the transfer to next generation sequencing works so badly. More and more people will look for applications with MBD proteins to actually get better results, less expensive, for next generation sequencing data.”
MBD Gets Kitted
Unlike MeDIP, there hasn’t been a ton of validated protocols, reagents or materials out there that don’t require a good amount of self-assembly and optimization…until recently. One company that has decided to hop aboard the MBD train, Invitrogen launched the MethylMiner™ Methylated DNA Enrichment Kit; their own tricked out version of the MCIp method that includes a variety of enhancements like the uber-strong binding between biotinylated MBD and the streptavidin MBD coated Dynal magnetic beads that researchers love, making the whole process pretty painless.
Post-doctoral Fellow, Dr. Nick Wong of the Murdoch Children’s Research Institute, is one of the early researchers to make the switch. “I’m seeing better consistency between replicates in the MBD approach as opposed to the MeDIP enrichment. Another improvement is the use of magnetic beads for DNA capture as opposed to agarose-G beads found in most MeDIP approaches,” added Wong.
Whether you’ve been on MeDIP for a while and are looking to explore new dimensions, or you’re just getting ready to take the dive into genome-wide methylation studies, you’ll definitely want to take a closer look at MBD-based enrichment.
*Despite using terms like “we’re betting” occasionally, EpiGenie does not endorse betting or gambling of any sort. Gambling can be a serious problem and should not be taken lightly, particularly when you lose the money that should go to a scientific website overhaul or a 88-1 long shot trifecta….wait, what were we talking about again?