K562 genomic DNA was bisulfite converted using the EpiTect Fast DNA Bisulfite Kit, and a 100 bp amplicon from the X-chromosome was amplified using cytosine-free primers to allow amplification of both unconverted and bisulfite converted DNA. PCR products were cloned into a vector and Sanger sequencing was done to identify converted cytosines. 1252 individual cytosines were analyzed, revealing a highly efficient cytosine conversion rate of 99.8%. This high conversion rate is especially significant as the genomic DNA was derived from a female; therefore, one of the 2 X-chromosomes is highly methylated.
Streamlined and straightforward, the EpiTect Fast 96 DNA Bisulfite Kit enables complete bisulfite conversion and cleanup of genomic DNA in approximately 1 hour.
Upon completing deparaffinization, lysis, and decrosslinking steps, the bisulfite conversion is completed using the included EpiTect Fast 96 DNA Bisulfite Kit.
Upon completion of the sample lysis steps, bisulfite conversion is completed using the included EpiTect Fast 96 DNA Bisulfite Kit.
Following bisulfite conversion with the EpiTect Fast DNA Bisulfite Kit, 1 µg of genomic DNA showed the lowest level of fragmentation, with a size distribution ranging from several hundred base pairs to over 6 kb, compared to other methods with peaks at 500 bp and 1 kb, respectively. Samples were analyzed on an Agilent RNA 6000 Nano Chip.
Following bisulfite conversion of genomic DNA using the EpiTect Fast DNA Bisulfite Kit or a kit from Supplier Z, unconverted genomic DNA and bisulfite converted DNA were coamplified and then sequenced by Pyrosequencing to quantify the rate of conversion of individual cytosines. [A] The EpiTect Fast DNA Bisulfite Kit gave comparable results to a kit from Supplier Z when 2 µg of DNA was analyzed. [B] However, when a limited amount of DNA was analyzed (10 ng), the 4 cytosine residues shown in this Pyrogram demonstrate that the EpiTect Fast DNA Bisulfite Kit delivered significantly better results than the kit from Supplier Z. (Percentage of each cytosine residue that did not undergo bisulfite conversion using the kit from Supplier Z vs. the EpiTect Fast DNA Bisulfite Kit: 12% vs. 2%, 5% vs. 1%, 37% vs. 9% and 11% vs. 2%, respectively.)
Upon addition to the bisulfite conversion reaction, the DNA Protect Buffer should turn from green to blue, indicating sufficient mixing and correct pH.
Depending on sample type, the bisulfite conversion of DNA calls for the use of a specific kit and protocol. Kits contain buffers optimized for DNA extraction and all reagents for the DNA bisulfite conversion.