When Aristotle compared epigenetics to a net (1), he could not have predicted how right he was. Recent research has revealed that mechanisms underlying epigenetic effects are numerous and interdependent as are the knots in a net. Each epigenetic mechanism has its players: enzymes, functional groups, substrates etc. The most important aspect of an epigenetic trait is its reversibility. Methylation of DNA was the first epigenetic modification to be discovered, and 5-cytosine methylation was the first to be linked with gene expression status. Currently, the most popular method for measuring CpG island methylation status is a bisulfite treatment of DNA followed by PCR or sequencing.
In this week’s webinar, Promega R&D scientist, Karen Reece focused on a workflow from DNA purification to analysis. She described the best methods for DNA isolation, quantification, bisulfite conversion, PCR and sequencing.
“Start with High Quality Genomic DNA Free of Contaminants”
Because the deamination step during bisulfite treatment will destroy and fragment DNA, a researcher should start with sufficient quantities of DNA. Purity is equally important. DNA should be free of protein carryover and of certain size for successful downstream analyses. These criteria can be hard to meet as many clinical sample types, [e.g., formalin-fixed paraffin-embedded (FFPE) tissues] give low yield and degraded DNA. Thus, Karen devoted a significant portion of her webinar describing DNA isolation methods. There are specific kits for blood, cells or FFPE samples. Time spent for each method is important. Karen guided us through manual and automated DNA purification methods. You can refer to Karen’s webinar to determine which method is the best for your sample type and how to use proper controls. If your sample type is not described Promega’s Technical Service Scientists are always here to assist.
DNA quantification an important but underappreciated step. As Karen warned, “Performing a quantification step will help you get the best results from your downstream methods and assist in troubleshooting any issues that arise, which will save you time in the long run.” She recommended using UV absorbance or ssDNA dye-based quantitation. Each DNA quantitation method has inherent advantages and disadvantages. For example, DNA quantitation based on absorbance provides an indication of contaminant carry-over, whereas quantitation using fluorescent dyes confers better sensitivity. You can estimate DNA degradation by simple agarose gel electrophoresis or with an Agilent BioAnalyzer. The latter also quantifies DNA. However, the instrument is very costly.
Bisulfite Conversion
The harsh chemistry of bisulfite treatment fragments genomic DNA (gDNA). Temperature, pH and time all affect the degree of DNA fragmentation. In her webinar Karen mentions which bisulfite conversion kit works best and explained how to perform quality checks for DNA fragmentation before proceeding with downstream assays, as significant fragmentation may decrease recovery yield after conversion cleanup. For this reason it is good to assess levels of fragmentation using gel analysis or the BioAnalyzer.
Using bisulfite-specific PCR Testing to Assess Conversion Efficiency
Researchers should check for incomplete conversion, which can result in false positive calls of methylated Cs. Remember, bisulfite treatmen twill only convert ssDNA, so complex DNA that does not separate well into single molecules during denaturation may not be converted efficiently. Test the conversion efficiency using bisulfite-specific PCR. Amplify converted and unconverted DNA with a combination of wildtype- and converted-sequence-specific PCR primer pairs. Include pre-qualified converted and unconverted DNAs as positive and negative controls, respectively to ensure specific amplification.
Another obstacle associated with this method is PCR bias (2), which refers to preferential amplification of bisulfite-converted unmethylated DNA over bisulfite-converted methylated DNA. Common techniques to overcome this bias are based on destabilizing GC rich regions and secondary structure of methylated DNA. However there is no universal approach reported so far to overcome such problems. For an example of a modified PCR cycling profile that was used to correct PCR bias, please visit the archived webinar.
Sequencing and Microarrays
The final part of this webinar was devoted to describing powerful tools that scientists use to study gene expression by determining the methylation status of CpG islands. Gene-specific sequencing, next-generation sequencing, Illumina’s Infinium Assay, methylated DNA immunoprecipitation analyzed by microarray (MeDIP-chip), comprehensive high-throughput arrays for relative methylation (CHARM), and HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) differ in cost, complexity and resolution.
Karen’s webinar provided an overview of methods used to analyze cytosine methylation, one of the many DNA modifications associated with epigenetic phenomena.
- Athanasios S. Tsaftaris, Alexios N. Polidoros, Aliki Kapazoglou, Eleni Tani and Nives M. Kovačević (2008). Epigenetics and Plant Breeding. In Plant Breeding Reviews. Ed Jules Janick
- Moskalev, et al. Nucleic Acids Research, (2011) Correction of PCR-bias in quantitative DNA methylation studies by means of cubic polynomial regression Vol. 39, No. 11 e77
Nives Kovacevic
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Nicely done, Nina! Another good paper I found on the subject of PCR bias correction was the following: Nucl. Acids Res. (1997) 25 (21): 4422-4426.