MethylDetector™

DNA methylation is a naturally occurring event that affects cell function by altering gene expression. In methylation, a methyl group is added to the fifth-carbon of cytosine in a CpG dinucleotide by DNA methyltransferase. As aberrant methylation is prevalent in many human cancers, and because methylation is also involved in embryonic development and cell cycle regulation, much research depends on accurately quantifying DNA methylation. Many DNA methylation analysis methods begin by using bisulfite to convert unmethylated cytosines to uracils.^1,2 During conversion, methylated cytosines remain unchanged. The DNA is then amplified by PCR and analyzed by sequencing or restriction digest. A methylation profile of the sample can then be created by comparing the sequence of the converted DNA to untreated DNA. However, bisulfite conversion can be technically challenging, and it is desirable to confirm that the process was successful before spending time and money on sample analysis. To help ensure your success, the MethylDetector Kit provides optimized conversion reagents, an easy-to-use protocol and positive control PCR primers that are specific for bisulfite-converted DNA. Because these primers produce PCR product only if conversion has occurred, you can confirm the procedure worked before starting sequencing or other analysis methods.

Figure 1: Agarose gel analysis of PCR products generated with MethylDetector.

Three different DNA conversions were performed (Lanes: 1-3) and compared to an unconverted DNA control (Lane: 5) and to a no DNA control (Lane: 4). The presence of PCR product in only the converted samples demonstrates the conversion efficiency and reproducibility of the MethylDetector Kit.

Why use MethylDetector™?

• Works efficiently with high G/C content sequences and uncut DNA
• Reproducible assay consistently provides 99% conversion efficiency of unmethylated cytosines
• Optimized reagents and protocol with proven human controls
• Combined thermal denaturation and conversion reaction eliminates NaOH-mediated denaturation and streamlines procedure
• DNA purification columns eliminate the need for precipitation and a separate desulfonation step
• High yield of converted DNA is ideal for downstream analysis

The MethylDetector™ advantage

In the MethylDetector method, DNA of interest is rapidly heat denatured in a thermocycler in the presence of the bisulfite conversion reagent. The temperature is then lowered and the conversion reaction is performed. Unlike other methods, MethylDetector does not require an initial acid denaturation step as the conversion reagent includes a DNA denaturant, which saves you time and effort. After DNA conversion, the sample is added to the included DNA purification columns, and a simple, on-column desulfonation is performed. Ready-to-use DNA is then eluted from the columns. For your convenience, the included positive control PCR primers can be used to assess the success of the bisulfite conversion before you spend time and money on DNA sequencing. This is because the included primers only anneal to converted human DNA (Figure 1).

To read a product review from Dr. Fijalkowska from Johns Hopkins University click here.

References

1. Frommer, M. et al. (1992) PNAS. 89: 1827.
2. Clark, S.J. et al. (1994) Nuc. Acids Res. 22: 2990-2997.

Contents & Storage

Conversion Reagent, Denaturation Reagent, Hydroquinone, DNA purification columns and collection tubes, DNA Binding, Wash and Elution Buffers, Positive control PCR primers and 10X PCR Buffer. Store at 4°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.