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Methylation Sensitive Mobility Shift Assay
MSMSA is a DNA fragment analysis application used to assess the degree of methylation present in a given amplicon.
Samples from a fragment analysis assay can also be used in a downstream sequencing step, making this application ideal for screening. The assay primers are designed in regions that don’t contain CpGs using Methyl Primer Express® Software, so that PCR amplification is not dependent on methylation status.
Figure 1: Cumulative Estimation for Methylation State of CpG Islands within an Amplicon. Methylated fragments migrate faster (Panel A, B) than those that are unmethylated (Panel C).
Step-by-Step Guide to 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. Recommended Products: DNA Isolation |
 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. Recommended Products: Perform Bisulfate ConversionWhich Kits are Right for You? |
 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. Recommended Products: Perform PCR |
 Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis. Recommended Products: Prepare Sample for AnalysisSizing InformationTo learn more about our GeneScan Size standards used in sizing experiments please see our table below.
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 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. Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You? |
 Analyze the MSMSA profile. Which Data Analysis Software Is Right for You? |
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. Recommended Products: DNA Isolation |
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. Recommended Products: Perform Bisulfate ConversionWhich Kits are Right for You? |
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. Recommended Products: Perform PCR |
Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis. Recommended Products: Prepare Sample for AnalysisSizing InformationTo learn more about our GeneScan Size standards used in sizing experiments please see our table below.
|
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. Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You? |
Analyze the MSMSA profile. Which Data Analysis Software Is Right for You? |
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