Methylation Analysis by Sanger Sequencing
DNA methylation is involved in the regulation of many cellular processes, including X chromosome inactivation, chromosome stability, chromatin structure, embryonic development, and transcription. Bisulfite sequencing is the gold standard for validating methylation experiments, as only sequencing provides direct detection of methylation events as well as information across the entire region. One of the key steps in this process, bisulfite DNA conversion, allows precise analysis of methylation in the target region by converting all non-methylated cytosines into uracils (methylated cytosines remain unchanged).
Two sequencing workflows can be use to determine the methylation status:
- Bisulfite-Specific, PCR-Based Sequencing:
This application is used to identify which CpG dinucleotides are important for the biological process under investigation. The researcher performs direct PCR sequencing of a pool of fragments to determine (qualitatively) which CpG dinucleotide has changed.
- Bisulfite-Specific, Cloning-Based Sequencing
In this application, a population of fragments is segregated by cloning, providing quantitative results. Analysis is aimed at calculating the percentage of methylation observed for specific CpG dinucleotides in a candidate region.
Step-by-Step Guide to Methylation Analysis
DNA extraction is a critical first step in the experimental workflow of DNA sequencing-based methylation 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
Bisulfite Conversion Kits convert non-methylated cytosines (C) in DNA samples 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 Bisulfite Conversion
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. Amplification products of bisulfite-converted DNA can be analyzed by fragment analysis or sequencing.
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.Learn more
Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You?
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