The BisulFlash™ DNA Modification Kit is a complete set of optimized buffers and reagents to perform DNA modification using a next generation DNA bisulfite conversion technology developed by Epigentek. Through a proprietary composition which allows DNA denaturation and bisulfite conversion to be processed at the same time， the complete procedure is reduced to only 30 minutes. Furthermore， it prevents more than 90% of DNA loss， completely converting unmethylated cytosine into uracil.
Perfecting Bisulfite Conversion Traditional methods involve a separate denaturation step followed by a subsequent sodium bisulfite DNA conversion step -- but with the BisulFlash™ method， DNA denaturation status is concurrently sustained throughout the entire bisulfite DNA conversion process. This breakthrough approach enables the DNA conversion process to be sigificantly faster with higher conversion efficiency and accuracy. We continue to innovate with the development of the new BisulFlash™ kit by identifying four critical components of bisulfite conversion:
Speed: Reduce the entire procedure to as short as 30 minutes without any reagent setup time.
Efficiency: Completely convert unmethylated cytosine into uracil -- modified DNA > 99.99%.
DNA Protection: Protect against DNA degradation of which more than 90% of DNA loss can be prevented， allowing for greater recovery.
Sensitivity: Start with the lowest amount of input DNA for modification -- only 0.2 ng or just 50 cells.
The convenient ready-to-use， DNA conversion mix solution and single temperature incubation along with the features mentioned above allow for true perfection in bisulfite conversion. The BisulFlash™ DNA Modification Kit is suitable for bisulfite sequencing， MS-PCR， real time MS-PCR， and methylation microarray. Comparative Overview of Commercial Kits Data was obtained through actual use of the kit， customer feedback， or information provided by the supplier's datasheet or website.
Correct/Error Conversion Ratio
Minimum Starting DNA
Fig. 1. Schematic procedure of the BisulFlash™ DNA Modification Kit to obtain converted DNA.
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 convesion 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.
Rapid Results 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 posible.
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 (>99.999%)， 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-tranded 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. 2.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&trade kit treated DNA was completely converted， and no unconverted DNA in the treated samples was determined after 45 cycles.
Fig. 3.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.
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