Principle & Procedure
As a next generation bisulfite conversion tool, the BisulFlash™ DNA Modification Kit contains all reagents required for an ultra-fast bisulfite conversion on a DNA sample. With the ready-to-use conversion mix solution, DNA denaturation status is sustained throughout the entire bisulfite DNA conversion process. This proprietary solution allows the bisulfite reagents to rapidly convert all cytosine to uracil with negligible methylcytosine conversion. The unique DNA protection reagents contained in the mix can prevent the chemical and thermophilic degradation of DNA in the bisulfite treatment. The non-toxic DNA capture solution enables DNA to tightly bind to the column filter, thus DNA cleaning can be carried out on the column to effectively remove residual bisulfite and salts.
Only 30 minutes are required for the entire BisulFlash™ procedure -- from converting your sample DNA to pure bisulfite modified DNA. That is significantly faster than any currently used kits (2-8 h) or homebrew methods (>16 h). The BisulFlash™ kit provides everything required for a successful bisulfite conversion and DNA cleanup in the shortest time with the fewest steps possible.
Perfect DNA Conversion
Each reaction with the BisulFlash™ kit can use 0.2 ng – 1 µg of DNA. For optimal conversion, the DNA amount is 200-500 ng. The novel procedure and proprietary ready-to-use DNA conversion mix solution allow DNA denaturation and bisulfite conversion to occur at the same time and enable all cytosines to be converted to uracil (>99.99%), while 5-methylcytosine remains the same. The highly efficient cytosine conversion is proven by the low CT values obtained when amplifying converted DNA using real-time PCR (Fig. 2). This perfect conversion rate is superior to other bisulfite kits available on the market and provides repeatable and dependable downstream analysis.
Powerful DNA Protection
DNA protection reagents are added into the DNA conversion mix solution to prevent DNA from chemical and thermophilic degradation in the bisulfite treatment and provide effective DNA denaturation, resulting in single-stranded DNA necessary for complete cytosine conversion. The prevention of DNA degradation enables subsequent amplification and analysis of large PCR fragments (Fig. 3). The efficient integrated DNA cleanup and unique elution buffer allow for long-term storage (> 6 months) and multiple freezing/thawing of the converted DNA without affecting the DNA quality.
Fig. 3. Complete Cytosine Conversion: 200 ng of genomic DNA isolated from 3 cancer cell lines was treated with the BisulFlash™ DNA Modification Kit. Next, the unconverted and converted DNA in each treated sample were determined using unconverted DNA-specific and converted DNA-specific primers (β-actin, 110 bps), respectively. A: real time PCR; B: end-point PCR. The BisulFlash™ kit treated DNA was completely converted, and no unconverted DNA in the treated samples was determined after 45 cycles.
Fig. 4. Effective DNA Protection: Fully methylated human genomic DNA at various amounts (0.2 ng-200 ng) were converted using the BisulFlash™ DNA Modification Kit. 1 µl of 20 µl eluate was used for real time qPCR and a pair of primers was used to amplify converted DNA. As little as 0.2 ng DNA is sufficient for bisulfite conversion using the BisulFlash™ DNA Modification Kit. A: real time PCR; B: starting DNA amount-CT value curve.
BF1 (Conversion Mix Solution)
BF2 (Capture Solution)
BF3 (Desulphonation Solution)
BF4 (Elution Solution)
BF5 (Conversion Enhancer)
BF6 (Denaturation Enhancer)