Since the advent of bisulfite conversion over 15 years ago, dozens of labs have streamlined the procedure, new patents have been filed, and numerous research kits with “new and improved” versions of the protocol have been launched, yet the core principal of the method remains the same: change a C to a U. Over the years a number of downstream applications have been developed and introduced to analyze this simple base change, all driven by the same premise that is behind much of innovation: the current method isn’t cutting it.
MSP, MS-SnuPE, COBRA, MethylLight, MALDI-TOF, and pyrosequencing are all being used to analyze locus- and often multi-locus specific methylation in a variety of developmental samples and tumors. Moreover, many of these methods are being tweaked to work effectively with analysis technologies that weren’t available until recently, like high density microarrays and deep sequencing.Over the years we’ve seen many methods emerge, some because they address an unmet need, while others are making use of a lab’s capital purchase made for another project. Our friends at research tool companies have done their best to make sure their toys can be used for emerging applications too. The end result? A dozen different ways to get from C-U. Let’s take a look at the evolution of methods used to investigate DNA methylation.
Early DNA Methylation Analysis Methods
Some of the earliest attempts at studying methylation status of specific loci used methyl-sensitive enzyme (unable to cut methylated DNA) digestion to differentiate methylated from unmethylated DNA. When combined with Southern hybridizations, researchers could get an overall sense of the methylation state of a specific locus. This approach worked fine except it required a considerable amount of intact DNA, something that is rare in many useful samples (e.g formalin fixed paraffin embedded) samples, and it could only detect methylation differences at loci where the enzymes had recognition sites.
The lack of sensitivity of this approach was later addressed by using PCR, but the issue of enzyme recognition sites was still there. Moreover, with the increase in sensitivity came an increased risk of false positives when samples weren’t completely digested. Although useful in some research scenarios, the shortcomings of methyl-sensitive enzymatic digestion began to mount.
Meanwhile in Australia, researcher Marianne Frommer and colleagues were addressing some of the shortcomings of early genomic sequencing approaches methylation profiling by combining a new sample preparation approach, sodium bisulfite treatment of DNA, with sequencing.1 It wasn’t the easiest protocol, but bisulfite sequencing was certainly a step up in many respects. Different versions of bisulfite sequencing are still frequently used today and they still provide the only means to quantitate methylation at the level of the individual nucleotide. ??Of equal importance as the introduction of the modified sequencing protocol, was the introduction bisulfite conversion itself as it laid the foundation for the emergence of many downstream analysis techniques.
Methylation Specific PCR: Bisulfite Discovers its Sensitive Side
In 1996 James Herman and colleagues at Johns Hopkins combined bisulfite conversion with PCR2 which reduced the incidence of false positives sometimes seen with incomplete enzymatic digestion and eliminated the requirement that a recognition sites lie in the area of interest (Figure 2). Sensitive, easier, faster, and cheaper than bisulfite sequencing, methylation-specific PCR (MSP) provided a relatively compelling alternative that looked well suited for the clinic. MSP remains a widely used method today for quick, sensitive, and cost-effective locus-specific analysis, however, primer design isn’t trivial and results aren’t really that quantitative.
COBRA and SNuPE
Combined bisulfite restriction analysis (COBRA)3 and methylation-sensitive single nucleotide primer extension (Ms-SNuPE)4 were just two of several methods introduced after MSP. These methods, like MSP, were sensitive and could be applied to FFPE samples, but they enabled more quantitative analysis methylation than MSP. Despite having catchy names and some promising advantages, the protocols initially involved radioactivity and were somewhat laborious, limiting widespread adoption. These limitations could and would be addressed a decade later however (dramatic foreshadowing).
Let There Be MethylLight
Technological advances in the fields of PCR and sequencing would later pave the wave for the introduction of quantitative approaches. Quantitative methylation-specific PCR (QMSP), or MethylLight and methylation pyrosequencing, respectively, brought improved the ease of use, and enabled quantitative monitoring of methylation density.
MeDIP and McrBC: the Newcomers
Continuous innovation in sample preparation and downstream analysis methods has resulted in a diverse (and complicated) toolbox for methylation analysis. Despite the various new approaches that have been introduced over the years, bisulfite conversion remains the most prominent method for the first key step of differentiating between methylated and unmethylated populations, however, the importance of gaining an understanding of methylation at the genome-wide level has been a significant driver lead to alternatives to bisulfite conversion.
Two main alternatives have been gaining traction in recent years as alternatives to bisulfite conversion, however:1.) Immunoprecipitation with 5-Methyl Cytosine (MeDIP) 52.) Methyl-dependant enzyme digestion with McrBC 6 Both of these methods have been used successfully with microarrays, and qPCR. Genpathway, of San Diego, California combines DNA immunoprecipitation with a 5-methylcytosine antibody (MeDNA IP) and Affymetrix tiling microarrays to profile genome-wide methylation patterns as a service. Unlike previous groups using methyl-sensitive digestion, Orion Genomics, based in St. Louis, Missouri, has taken a slightly different approach to global methylation analysis.
By using McrBC, a robust, methylation-dependant restriction enzyme, to select for methylated DNA with high density microarrays, Orion has been able to identify a variety promising biomarkers. “The main problem with bisulphite array work is that the bisulphite turns a four base genome into a three base genome. This means that the complexity of the hybridization is reduced (for any hybridization based technology including PCR). Lowering the complexity means you make repeats…CTCTCTCTCT becomes TTTTTTTTTT etc. This makes hybridization based technologies a mess” explained Dr. Jeff Jeddeloh of Orion. “It’s possible that as read lengths improve, deep sequencing of bisulphite converted DNA might get around this issue…but right now it’s an assembly nightmare for the analysis.”