The Imprint® DNA Modification Kit provides the reagents needed for bisulfite conversion and post-modification clean-up of DNA samples in less than 2 hours. Once treated, sample DNA is ready for downstream analyses, such as bisulfite sequencing or methylation specific PCR (MSP), which allows for discrimination of methylated and unmethylated DNA.
It has been well demonstrated that DNA methylation plays an important role in the regulation of gene expression. At any given time about 70% of all CpG dinucleotides in the mammalian genome are methylated.1 Hypo- and hyper-methylation across the genome contribute to changes in gene expression. Determining the methylation status of DNA is an important tool in epigenetic studies.2,3
The Imprint® protocol allows the user to selectively perform DNA modification such that unmethylated cytosines are converted to uracil, while the methylated cytosines remain unchanged. For optimal modification the DNA quantity should be in the range of 50-200 ng, but DNA ranging from 0.1 ng to 1 µg has been used successfully. The modified DNA generated from the our Imprint® DNA Modification procedure is suitable for use in bisulfite sequencing, MSP, pyrosequencing and methylation microarrays.
All components can be stored at room temperature. Each vial of DNA Modification Powder is sufficient for ten DNA modifications. Once dissolved, the solution can be stored at -20 °C for one week, kept away from light. Before use, the frozen solution must be thawed at room temperature and vortexed for two minutes.
The Imprint® DNA Modification Kit contains all reagents required for the bisulfite conversion of DNA. DNA denaturation and bisulfite modification are carried out simultaneously. In the modification process, bisulfite reacts specifically with single-stranded DNA to deaminate the cytosine, creating a uracil residue. The unique DNA protection reagents in the modification buffer prevent chemical and thermophilic degradation of DNA in the bisulfite treatment. The Capture Solution enables the DNA to tightly bind to the column filter. This allows for the effective removal of residual sodium bisulfite and salts. The eluted modified DNA can be used immediately, or stored at –20 °C for up to 2 months.
The Imprint® protocol offers the flexibility of either a One-Step or a Two-Step Modification procedure. The One-Step Modification is included as a more convenient procedure to be used with higher DNA input (10 ng to 1 µg).The Two-Step Modification procedure is recommended for low DNA input (100 pg to 10 ng). A BSA solution using Catalog Number B8667 (20 mg/mL) diluted to 0.5 mg/mL (12.5 µL BSA to 487.5 µL water) can be used as a carrier. The BSA-water solution will improve DNA recovery with low input concentrations.
One-Step Modification Procedure
Two-Step Modification Procedure
Post Modification DNA Clean Up
Figure 1 Bisulfite modification was performed on decreasing concentrations of human genomic DNA using Imprint® MOD50 and kits from two other suppliers. Methylation specific PCR (MSP) was then performed on all samples using β-Actin gene specific primers (109 bp amplicon). For all suppliers lanes from left to right are 100 ng, 10 ng, 1 ng, 100 pg, 50 pg and no sample respectively. Suppliers C and D (not shown) have no product at 50 pg. These results demonstrate the superior sensitivity of the Imprint® kit relative to other suppliers.
What is the difference between the one-step DNA modification and the two-step DNA modification?
In the one-step DNA modification, DNA is denatured by heating, which allows DNA denaturation and bisulfite modification to be carried out simultaneously. Because one-step DNA modification may increase DNA degradation, the procedure is best suited for higher DNA input (10 ng to 1 µg). In the two-step DNA modification, DNA is denatured chemically followed by bisulfite treatment. This procedure is recommended for lower inputs (100 pg to 10 ng). Both kits are suitable for modification using DNA isolated from various sources.
How much starting DNA is required for DNA modification with this kit?
The starting DNA required for DNA modification can be as low as 50 pg. The signal of the modified DNA can be detected with real-time PCR.
Why is only 90 minutes required for DNA modification?
With our unique modification composition, 90 minutes is sufficient for more than 99% C-U conversion while DNA degradation is greatly prevented. We have observed that increase in modification time did not significantly increase C-U conversion, while yield of modified DNA was significantly reduced most likely due to increased DNA degradation.
How long can the modified DNA be stored?
The modified DNA generated with this kit can be stored for 2 months at –20 °C, 6 months at –80 °C.
Can the kit be used for modifying DNA isolated from formalin-fixed and paraffin-embedded (FFPE) samples?
Yes. However, it is important to note that some fixation protocols severely degrade DNA. Therefore we recommend the user attempt to amplify the gene of interest before bisulfite treatment. If the gene can be amplified, we recommend the two-step protocol for FFPE samples. The Imprint bisulfite modification kit includes DNA stabilizing reagents that minimize sample degradation.
This product is for research use only. It is not intended and should not be used for any human or animal therapeutic or diagnostic purposes. Any such unauthorized use will be in violation of proprietary rights of Epigentek Group Inc. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Imprint is a registered trademark of Merck KGaA, Darmstadt, Germany and/or its affiliates.