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EZ DNA Methylation™ Kit

Streamlined, proven procedure for bisulfite conversion of DNA.
Desulphonation and recovery of bisulfite-treated DNA with a spin column.
Recovered DNA is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.

Product Size Catalog # Price Qty
EZ DNA Methylation™ Kit 50 Rxns D5001
EZ DNA Methylation™ Kit 200 Rxns D5002

About EZ DNA Methylation™ Kit

The EZ DNA Methylation™ Kit features a simplified procedure that streamlines bisulfite treatment of DNA. The kit is based on the three-step reaction that takes place between cytosine and sodium bisulfite where cytosine is converted into uracil. The product's innovative in-column desulphonation technologies eliminate otherwise cumbersome precipitations. The kit is designed to reduce template degradation, minimize DNA loss during treatment and cleanup, while ensuring complete conversion of the DNA. Purified, converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion, sequencing, microarrays, etc. This kit is validated for use with Illumina's GoldenGate® and Infinium® Assays.

Learn More About Bisulfite Conversion Click to see which kit is right for you

Format Spin Column
DNA Recovery > 80%
Processing Time 12 - 16 hrs
Equipment Microcentrifuge
Binding Capacity 5 µg/prep
Elution Volume ≥ 10 µl
Conversion Efficiency > 99%
Input Plasmid, genomic, and endonuclease digested DNAs.

Conversion of non-methylated cytosine. A 459 bp PCR product amplified from either DNA converted with the EZ DNA Methylation™ Kit (lane 1) or from untreated DNA (lane 2) was digested with EcoR I. The cytosine at the EcoR I site was converted to uracil during treatment, preventing cleavage of the DNA by the endonuclease.






Step 1

Add 5 µl of M-Dilution Buffer to the DNA sample and adjust the total volume to 50 µl with water.  Mix the sample by flicking or pipetting up and down.

Example: For 14 µl of a DNA sample add 5 µl M-Dilution Buffer and 31 µl water. 

Step 2

Incubate the sample at 37°C for 15 minutes.

Step 3

After the above incubation, add 100 µl of the prepared CT Conversion Reagent to each sample and mix.

Step 4

Incubate the sample in the dark at 50°C for 12-16 hours.

Please see Appendix (page 6) for alternative incubation conditions (e.g., when using the Illumina Infinium® Methylation Assay)

Step 5

Incubate the sample at 0-4°C (e.g., on ice) for 10 minutes.

Step 6

Add 400 µl of M-Binding Buffer to a Zymo-Spin™ IC Column and place the column into a provided Collection Tube.

Step 7

Load the sample (from Step 5) into the Zymo-Spin™ IC Column containing the M-Binding Buffer.  Close the cap and mix by inverting the column several times.

Step 8

Centrifuge at full speed (≥10,000 x g) for 30 seconds.  Discard the flow-through.

Step 9

Add 100 µl of M-Wash Buffer to the column.  Centrifuge at full speed for 30 seconds.

Step 10

Add 200 µl of M-Desulphonation Buffer to the column and let stand at room temperature (20-30°C) for 15-20 minutes.  After the incubation, centrifuge at full speed for 30 seconds.

Step 11

Add 200 µl of M-Wash Buffer to the column.  Centrifuge at full speed for 30 seconds.  Add another 200 µl of M-Wash Buffer and centrifuge for an additional 30 seconds.

Step 12

Place the column into a 1.5 ml microcentrifuge tube.  Add 10 µl of M-Elution Buffer directly to the column matrix.  Centrifuge for 30 seconds at full speed to elute the DNA.

The DNA is ready for immediate analysis or can be stored at or below -20°C for later use.  For long term storage, store at or below -70°C.  We recommend using 1-4 µl of eluted DNA for each PCR, however, up to 10 µl can be used if necessary.  The elution volume can be < 10 µl depending on the requirements of your experiments, but small elution volumes will yield more concentrated DNA.

For specific notes and additional information, please see the product protocol PDF.
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Featured Citations

Researchers from Germany used the EZ DNA Methylation Kit™ to bisulfite convert genomic DNA from ex vivo isolated T cell subsets to study the changes in DNA methylation during differentiation of naïve T cells into Th cell subsets. Further methylome analysis through methylation sensitive high-resolution melting (MS-HRM) and pyrosequencing demonstrated that CD4+ T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions. Through this analysis, the researchers were able to identify a unique Th17-specific epigenetic signature and reinforce the finding that Th cell differentiation is associated with major epigenetic changes.
Researchers from Brazil studying bipolar disorder (BD), aimed to identify epigenetic mechanisms associated with the development and progression of BD. The EZ DNA Methylation Kit™ was used to bisulfite treat DNA samples from patients with BD, first degree relatives, and healthy controls, prior to DNA methylation analysis by pyro sequencing. Based on previous experiments of epigenetic mechanisms modulating the FKBP51 feed-back loop and evidence suggesting the FKBP5 gene plays a role in altered DNA methylation patterns, the researchers focused on regions of the FKBP5 gene during the DNA methylation analysis. Patients with BD presented with increased intronic methylation levels at the FKBP5 gene region which may be responsible for reduced mRNA expression that was previously demonstrated by the group. This is one of the first studies to look at epigenetic mechanisms of the FKBP5 gene in patients with BD and their first-degree relatives.
Researchers studying circulating unmethylated and methylated preproinsulin (INS) DNA in type 1 diabetes (T1D) sought to test the differences between subjects with new-onset T1D and controls. DNA was extracted from serum using the ZR Serum DNA Kit™ and bisulfite converted with the EZ DNA Methylation Kit™ or the EZ DNA Methylation-Lightning Kit™. Droplet digital PCR was used to measure unmethylated and methylated serum levels. The data demonstrated that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. The use of methylation levels as potential biomarkers for diabetes may prove to be a novel tool in the near future.
Chromatin from clear cell renal cell carcinoma patient tissues were treated with M.SssI to methylate CpGs in open chromatin regions. DNA was then isolated and bisulfite-converted using the EZ DNA Methylation Kit and submitted for HM450 analysis. M.SssI-treated/untreated DNA methylation values were then compared to simultaneously measure endogenous DNA methylation and chromatin accessibility in renal carcinoma and adjacent normal samples.
The EZ DNA Methylation™ Kit was used to bisulfite convert DNA to study the methylation levels of various genes related to the JAK-STAT signaling pathway in T-cell lymphoblastic lymphoma (T-LBL). The researchers used methylation specific PCR and sequencing to further elucidate the role of JAK2 and more specifically SOCS3, a suppressor of cytokine signaling in this disease. The data supports current literature demonstrating that SOCS3 is often hypermethylated in T-LBL patients and that these patients may benefit from treatment with DNA methylation inhibitors.
Researchers from New York investigated the role of changes in epigenome structure in age-related changes of gene expression and ovarian function. The EZ DNA Methylation™ Kit was used to bisulfite convert DNA purified from human ovarian granulosa cells. Next generation sequencing technologies such as RRBS further allowed the researchers to link epigenome structure to age-related physiology and pathology in female fertility.
This study investigated the effects of high-fat overfeeding in healthy low birth weight and normal birth weight individuals. Genomic DNA from both groups was extracted from blood samples and bisulfite treated using EZ DNA Methylation Kit. Bisulfite converted DNA was assessed using Illumina’s Infinium 27k BeadArray. Lower DNA methylation plasticity in skeletal muscle was found in the low birth weight group and this could potentially contribute to the understanding the link between low birth weight and increase risk of type 2 diabetes.
Researchers established and systematically evaluated a statistical method to predict methylation patterns in a target tissue using more easily accessed surrogate tissues. DNA was bisulfite converted using EZ DNA Methylation Kit and profiled using Illumina Infinium HumanMethylation27 and HumanMethylation450 arrays. The novel statistical model showed improved accuracy in predicting the methylation in the target tissue.
Researchers identified disease-associated DNA methylation differences for atopic dermatitis. DNA methylations patterns were investigated in whole blood, T cells, B cells, lesional, and non-lesional epidermis from both healthy and atopic dermatitis patients. Striking methylation differences were found between lesional epidermis and healthy control epidermis, opening the door to future investigations of epigenetic mechanisms in atopic dermatitis.
Researchers used the EZ DNA Methylation kit for bisulfite conversion of DNA extracted from pancreatic ductal adenocarcinoma tissues. Genome-wide DNA methylation patterns were analyzed to show that aberrant DNA methylation patterns are widespread in pancreatic cancer, particularly in cancer-signaling pathways known to be important in pancreatic carcinogenesis.
The authors investigated the association between human newborn neurobehaviour and placental leptin DNA methylation. Bisulfite pyrosequencing was employed using the bisulfite-treated DNA for leptin promoter methylation detection. Infant neurobehaviour was evaluated with the NICU Network Neurobehavioral Scales. Increased methylation of the placental leptin promotor was associated with a statistically significant neurobehavioral profile in male infants, suggesting a role in early neurodevelopement.
The EZ DNA Methylation™ Kit from Zymo Research was used by researchers to investigate DNA methylation changes in the development of osteoclast cells. Osteoclasts are derived from monocytes in the hematopoietic system, and these cells can degrade bones, which is an important step in the healing process of broken bones. The researchers observed that certain genes exhibited DNA hypermethylation, and other genes showed a decrease in DNA methylation during differentiation of monocytes to osteoclasts, leading them to conclude that DNA methylation is a key epigenetic mechanism that regulates this process.
In this publication, the authors provide a roadmap of modifications, strategies, and troubleshooting approaches to optimize sequencing of multiplexed libraries for Reduced Representation Bisulfite Sequencing (RRBS). Furthermore, the researchers found that several other published methods that recommended two rounds of bisulfite conversion with the Qiagen EpiTect kit for complete bisulfite conversion of human RRBS libraries resulted in significant loss of the template. The scientists achieved more consistent bisulfite conversion of size-selected libraries using the EZ DNA Methylation kit from Zymo Research. The use of the kit resulted in consistent conversion of human genomic DNA and minimal loss during the process.
In a study to determine how global DNA methylation patterns change with age, researchers used the EZ DNA Methylation Kit to bisulfite convert genomic DNA blood samples taken from newborn babies and 100+ year old patients. The authors subsequently subjected the bisulfite-converted DNA to whole genome bisulfite sequencing, 450 K CpG site microarray analysis, and bisulfite genomic sequencing and pyrosequencing, and they found that genome wide DNA methylation content significantly decreased with age.
In a genome-wide study investigating DNA methylation levels in acute myeloid leukemia cells, researchers used the EZ DNA MethylationTM Kit to bisulfite convert genomic DNA obtained from clinical samples. PCR amplification followed by bisulfite sequencing of the converted DNA helped to show that patients diagnosed with acute myeloid leukemia have distinct methylomes in their cancer cells compared to normal cells.
DNA from human colorectal tumors and normal adjacent tissues was bisulfite converted using Zymo’s EZ DNA Methylation Kit and converted DNA was analyzed by Next-Gen sequencing. Whole genome bisulfite sequencing revealed focal hypermethylation at CpG islands within regions of long-range hypomethylation and possible silencing programs directed by the three-dimensional structure of chromatin within the nucleus of cancer cells.

"I liked that the yield of DNA was excellent and I was able to get PCR product from cheek swabs samples that had been in the freezer for years. I had tried to get product with a competitors kit, but it didn't work, and was over-complicated."
Mary W. (DOGenes Inc.)
Click here to submit your review of the EZ DNA Methylation™ Kit.

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