Accurate and reliable methods to detect DNA methylation patterns are vital for researchers interested in the study of epigenetics. Understanding methylation profiles can be used for disease identification, from cancer to genetic conditions; as well as other important aspects of biology, including gene imprinting and embryonic development.
DNA methylation is a biochemical process involving the addition of a methyl group to the cytosine nucleotide via a methyltransferase enzyme. Nearly all methylation in the mammalian genome occurs in 5′-CpG-3′ sequences, but other sequence contexts can also be altered (1).
There are several ways to determine whether a gene contains methylated DNA, but the initial step in almost all methods involves bisulfite treatment. In this method, researchers take advantage of the fact that, when treated with bisulfite, non-methylated cytosines are converted to uracil, while methylcytosines remain unchanged (2).
Zymo Research Corporation’s EZ DNA Methylation™ Kits and standards provide a consolidated suite of proven bisulfite technologies for DNA treatment for methylation analysis. Highly trusted by the research community, these are the most cited bisulfite technologies in the literature (3).
With a conversion rate above 99%, the EZ DNA Methylation™ Kits offer the most reliable workflows currently available on the market for bisulfite conversion of methylated DNA. All kits are designed to minimize loss and ensure complete conversion of the DNA. This has become a particularly appealing system for researchers due to the use of the innovative Zymo-Spin™ column, eliminating heavy and complex precipitations and offering no buffer retention.
Converted and purified DNA obtained through any of the EZ DNA Methylation Kits can subsequently be analyzed by PCR amplification, COBRA, MSP or NGS to determine the methylation pattern.
Validated for use with GoldenGate® and Infinium® from Illumina, the EZ DNA Methylation™ Kit (D5001/D5002) features a streamlined bisulfite treatment of DNA. Spanish researchers relied on this three-step process, followed by whole genome bisulfite sequencing, 450 K CpG site microarray analysis, bisulfite genomic sequencing and pyrosequencing to determine how DNA methylation patterns change with age (4).
The highest selling product and most cited in the literature (3), however, is the EZ DNA Methylation Gold™ Kit (D5005/D5006). This kit combines DNA denaturation and bisulfite conversion into one single step, reducing processing time to only 4 hours without affecting results. Using this approach, a team of USA-based scientists bisulfite-treated genomic DNA from honeybees to compare DNA methylation levels between subclasses of worker bees for whole genome bisulfite sequencing (5).
For experiments requiring cells, blood or tissue as the direct input, without the prerequisite of DNA purification, the best system is the EZ DNA Methylation Direct™ Kits (D5020/D5021). In addition to a fast processing time (around 4 hours), this kit boasts an increased sensitivity, requiring as little as 10 cells or 50 pg DNA. Similarly to the EZ DNA methylation Gold kits, DNA denaturation and bisulfite conversion are joined into a single step. This was the kit chosen by researchers from Harvard Medical School to study methylation patterns in breast cancer cells, followed by methylation-specific PCR (MSP) assays (6).
Finally, with pre-prepared reagent, the fastest processing time, and high conversion efficiency, the EZ DNA Methylation Lighting Kit™ (D5030/D5031) is ideal for highly sensitive workflows and automation. The key to this method is the ready-to-use conversion reagent which reduces the conversion time to only 1 hour. Also from Harvard, a team of geneticists used the EZ DNA Methylation Lighting Kit to generate bisulfite sequencing libraries to compare methylation levels of mutated and wild type mouse primordial germ cells (PGCs). The authors successfully obtained full DNA conversion, eliminating the need for post-mapping filtering (7).
All EZ DNA Methylation™ Kits can be combined with DNA standards, to serve as controls and measure the efficiency of DNA conversion. Methylated and Non-Methylated DNA standards (D5010, D5011, D5012, D5014 and D5017) are appropriate for any kit, as positive and negative controls. However, especially for downstream analysis involving amplification assays, Zymo Research Corporation has developed the Bisulfite-converted Universal Methylated Human DNA Standard (D5015).
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- Frommer M. Proc. Natl. Acad. Sci. USA. 1992; 89(5): 1827-1831.
- Hernández H, Tse MY, Pang SC, Arboleda H and Forero DA (2013). Optimizing methodologies for PCR-based DNA methylation analysis. BioTechniques, 55: 181–197
- Heyn H, Li N, Ferreira HJ, et al. (2012) Distinct DNA methylomes of newborns and centenarians. Proc Natl Acad Sci U S A. 109(26):10522-7
- Herb, B.R., et al. (2012). Reversible switching between epigenetic states in honeybee behavioral subcastes. Nature Neurosci, 15(10): 1371-1373.
- Song SJ, et al. (2013) MicroRNA-Antagonism Regulates Breast Cancer Stemness and Metastasis via TET-Family-Dependent Chromatin Remodeling. Cell, 154 (2), 311-24
- Yamaguchi, S., et al. (2012) Tet1 controls meiosis by regulating meiotic gene expression. Nature 492: 443-447
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