Methylation Sensitive Mobility Shift Assay

DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.

The methylSEQr™ Bisulfite Conversion Kit converts non-methylated cytosines (C) in the DNA sample to uracil (U). Methylated cytosines remain unchanged. Comparing the sequences of treated vs. non-treated DNA and detection of converted cytosines (now apparent as T base) determine which cytosines in the sample are methylated in CpG dinucleotides.

Design high quality PCR primers for methylation mapping experiments. Simply cut and paste in your region of interest in the primer design software. The tool searches for CpG islands and simulates bisulfite modification of DNA in silico. Perform PCR to amplify region of interest.

Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis.

During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system.

Analyze the MSMSA profile.