The OneStep qMethyl™ Kit from Zymo Research provides a simple, straightforward, and bisulfite-free procedure for rapid, locus-specific DNA methylation assessment. Simply add DNA into the appropriate reaction mix then quantitate via real-time PCR OneStep!
|Format||Provided Test Reaction PreMix and Reference Reaction PreMix contain SYTO®9 dye. Kit can be purchased without dye in the premixes (see OneStep qMethyl™ -Lite)|
|Processing Time||4.5 hrs|
|Input||High quality DNA suitable for use in restriction enzyme digestion. Require A260/280 > 1.8, A260/230 > 1.8.|
|Storage||Store at -20°C for up to 12 months. Avoid repeated freeze/thawing of reagents. Prolong storage at -80°C. 2X Test Reaction PreMix and 2X Reference Reaction PreMix are light sensitive, minimize light exposure.|
Simple, One Step DNA Methylation Determination
Unlike conventional qAMP procedures that contain multiple steps, the OneStep qMethyl™ method integrates the workflow into a simple, single step reaction. This minimizes errors that can occur during setup and the likelihood of contamination while allowing for rapid and accurate DNA methylation level detection.
The OneStep qMethyl™ Kit is used for the detection of locus-specific DNA methylation via the selective amplification of a methylated region of DNA. This is accomplished by splitting any DNA to be tested into two parts: a "Test Reaction" and a "Reference Reaction" (see Figure 1 below). DNA in the Test Reaction is digested with Methylation Sensitive Restriction Enzymes (MSREs) while DNA in the Reference Reaction is not. The DNA from both samples is then amplified using real-time PCR in the presence of SYTO®9 fluorescent dye and then quantitated.
Schematics A and B (above) illustrate the sample workflow of Non-methylated DNA and Methylated DNAs. In both cases the DNA is divided in two parts; a Test Reaction and a Reference Reaction. Test Reaction samples are MSRE digested while the Reference Reaction samples are not (mock digested). Following digestion, DNA from both samples is used for real-time PCR. The white lollipops in the image represent unmethylated cytosines and black lollipops methylated cytosines in CpG dinucleotide context. Following real-time PCR, amplification plots (C and D) demonstrate non-methylated DNA exhibits large differences in the Ct values for Test and Reference Reactions (C) while highly methylated DNA samples exhibit little difference (D).