EZ DNA Methylation-Direct™ Kit

Complete bisulfite conversion of DNA directly from blood, soft tissue, cells, FFPE samples, and LCM samples.
Compatible with small sample inputs "“ as few as 10 cells or 50 pg DNA.
Desulfonation and recovery of bisulfite-treated DNA with a spin column.

Product Size Catalog # Price Qty
EZ DNA Methylation-Direct™ Kit 50 Rxns D5020
$193.00
EZ DNA Methylation-Direct™ Kit 200 Rxns D5021
$547.00

About EZ DNA Methylation-Direct™ Kit

The EZ DNA Methylation-Direct™ Kit is a further refinement of our popular EZ DNA Methylation™ and EZ DNA Methylation-Gold™ kits. These products feature reliable and complete DNA bisulfite conversion directly from blood, tissue, and cells without the prerequisite for DNA purification. The increased sensitivity of these kits make it possible to amplify bisulfite-converted DNA from as few as 10 cells or 50 pg DNA. Like the EZ DNA Methylation-Gold™ kits, DNA denaturation and bisulfite conversion processes are combined into a single step. Recovered bisulfite-converted DNA is ideal for PCR amplification for downstream analyses including restriction endonuclease digestion, sequencing, microarrays, etc.

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


Format Spin Column
DNA Recovery > 80%
Processing Time 4 hrs
Equipment Microcentrifuge
Binding Capacity 5 µg/prep
Elution Volume ≥ 10 µl
Conversion Efficiency > 99.5%
Input DNA, cells, blood, tissue, FFPE

 

EZ DNA Methylation-Direct™ Kit bisulfite chemistry significantly improves C to U conversion kinetics. DNA was converted using either EZ DNA Methylation-Direct™ or conventional bisulfite chemistries. Recovered DNA was amplified by PCR, then cloned. Sequences from individual clones were analyzed and quantitated. These data show that EZ DNA Methylation-Direct™ bisulfite chemistry improves the rate and extent (< 99.8%) of C to U conversion of DNA as compared to conventional bisulfite chemistry.

 

Either blood, tissue, cells, or purified DNA can be used as the starting material for the EZ DNA Methylation-Direct™ Kit. If purified DNA is used, then proceed directly to Section II.

Section I: Sample Digestion with Proteinase K

1. Set up Proteinase K digestions for your specific sample type (please see detailed protocol for more information).

2. Incubate the sample(s) for 20 minutes at 50°C.

Section II. Bisulfite Conversion of DNA

1. Add 20 μl of sample from Step 3 (Section I) to 130 μl of CT Conversion Reagent solution in a PCR tube.

2. Place the PCR tube(s) in a thermal cycler and perform the following steps:

1. 98°C for 8 minutes

2. 64°C for 3.5 hours

3. 4°C storage for up to 20 hours

3. Add 600 μl of M-Binding Buffer into a Zymo-Spin™ IC Column and place the column into a provided Collection Tube.

4. Load the sample (from Step 2) into the Zymo-Spin™ IC Column containing the M-Binding Buffer. Close the cap and mix by inverting the column several times.

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

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

7. 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.

8. 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.

9. 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

Bisulfite treatment of DNA from fibroblast cells of Aicardi-Goutières syndrome (AGS) patients was performed using the EZ DNA Methylation-Direct™ Kit prior to MethylC-seq. Results showed a global loss of DNA methylation from patients with TREX1, RNASEH2, and SAMHD1 mutations, indicating epigenetic abnormalities in AGS.
In this publication, researchers investigated the regulatory effects of BRCA and glucocorticoid receptor (GR). Genomic DNA extracted from both ovarian cancer and normal ovarian tissue was bisulfite converted using EZ DNA Methylation-Direct Kit and BRCA1 promoter methylation was analyzed. Immunochemistry, real-time PCR, regression analysis, knockdown, and overexpression experiments were also performed to examine the relationship between BRCA1 and GR expression levels, which were found to positively correlate in cancer tissues.
Epigenetic changes were shown to be induced in the midbrain of adult mice by social isolation from 3-6 months of age. Genomic DNA was isolated from the midbrain of the “lonely” mice and was bisulfite converted using the EZ DNA Methylation-Direct Kit. Bisulfite pyrosequencing was performed and the researchers found that social isolation of adult male C57BL/6 mice led to global DNA methylation changes in the midbrain.
The EZ DNA Methylation-Direct Kit was used by researchers to bisulfite convert DNA from breast cancer cell lines and patient tissues prior to performing methylation-specific PCR (MSP) and pyrosequencing. They found that DNA methylation is involved in the regulation of heparanase during breast cancer progression and significantly correlated with clinical stage.
Researchers extracted DNA from in vitro differentiated mouse and human embryonic stem cells, the DNA was used for both mDIP microarray analysis and bisulfite deep sequencing. They show that ES cell lines and cells derived from them are subject to significant aberrant CpG island de novo methylation which is exacerbated by differentiation in vitro and may inhibit normal differentiation.
Researchers showed that DNA methylation is involved in the regulation of TMPRSS2, a downstream androgen receptor signaling gene important in prostate cancer. Prostate cancer cells were subjected to a bisulfite conversion using EZ DNA Methylation-Direct Kit and methylation levels were analyzed. The data suggest that high levels of DNMT1 lead to the transcriptional repression of TMPRSS2 in AR-negative prostate cancer cells.
Researchers at Harvard Medical School used the EZ DNA Methylation-Direct™ Kit to bisulfite convert DNA in breast cancer studies prior to performing methylation-specific PCR (MSP) assays. They found that overexpression of miR-22 epigenetically silenced the expression of another miRNA, miR-200, by targeting TET enzymes – leading to increased DNA methylation at the miR-200 promoter.

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