[A] DNA Protect Buffer prevents DNA fragmentation during bisulfite treatment of DNA and facilitates the formation of single-stranded DNA, enabling complete bisulfite conversion. [B] The pH indicator dye in the DNA Protect Buffer enables visualization of complete reaction mixing, thereby ensuring the correct pH is achieved for complete cytosine conversion.
DNA (1 µg) isolated from an FFPE sample using the QIAamp DNA Mini Kit was converted with the EpiTect Bisulfite Kit using the FFPE protocol. 1 μl of the 40 μl eluate was analyzed by [A] end-point PCR and [B] TaqMan probe-based real-time PCR. Both assays for the Glutathione S-Transferase gene, were specific for converted DNA. In each application the unconverted genomic DNA (control) was not amplified.
The complete EpiTect Bisulfite Kit centrifugation or vacuum procedure — from DNA to pure bisulfite converted DNA ready for analysis — takes less than 6 hours. Traditional bisulfite conversion procedures can take over 18 hours and require extensive user interaction.
Over 900 clones generated by PCR from 6 independent EpiTect Bisulfite reactions (1 μg DNA each) and cloned using the QIAGEN PCR Cloning Kit were sequenced. Over 150,000 of the 450,000 sequenced nucleotides were unmethylated cytosine residues (DNA 1) that were converted into uracil residues (DNA 2). Cytosine to uracil conversion rates of 99.4–99.8% were obtained. The sequence shown is a small portion of the total sequence analyzed. DNA 1: untreated DNA; DNA 2: converted DNA.
A 1 μg aliquot of genomic DNA was converted using the EpiTect Bisulfite Kit or bisulfite kits from alternative suppliers (Supplier E and Supplier Z) according to the manufacturer's instructions. Next, the presence of unconverted DNA in comparable amounts of each sample was determined by [A] end-point PCR using the HotStarTaq Plus Master Mix Kit and [B] SYBR® Green based real-time PCR using the QuantiTect SYBR® Green PCR Kit. The EpiTect Bisulfite Kit treated DNA was completely converted, while DNA treated using kits from Suppliers E and Z contained a significant proportion of unconverted DNA.
Human genomic DNA was purified from blood using the QIAamp DNA Blood Mini Kit, and various amounts (1 ng – 1 µg) were converted using the EpiTect Bisulfite Kit. PCR was performed using the HotStarTaq Master Mix Kit and 2 sets of primers designed to amplify converted DNA. A 5 µl aliquot of each PCR was loaded onto a 1.3% agarose gel. As little as 1 ng DNA is sufficient for conversion using the EpiTect Bisulfite Kit. C: untreated genomic DNA (negative control). M: marker.
Bisulfite converted DNA generated with EpiTect Kits was stored at –20°C for up to 36 months, and amplified at several time intervals. The resulting threshold cycle values at all time points for various DNA amounts were comparable to those obtained with freshly converted DNA (time point 0).