Methylation levels are measured using various techniques, each with its own advantages and disadvantages. Here are some common methods:
1. Bisulfite Sequencing
- Principle: Bisulfite sequencing converts unmethylated cytosines to uracil, while methylated cytosines remain unchanged. This allows for the differentiation of methylated and unmethylated cytosines by sequencing the converted DNA.
- Advantages: High accuracy and resolution, can identify specific methylation patterns.
- Disadvantages: Expensive, time-consuming, and requires specialized equipment.
2. Methylation-Specific PCR (MSP)
- Principle: MSP uses primers that specifically bind to either methylated or unmethylated DNA sequences. Amplification of the target sequence indicates the presence of methylation or lack thereof.
- Advantages: Relatively inexpensive, quick, and easy to perform.
- Disadvantages: Limited resolution, only detects methylation at specific sites.
3. Pyrosequencing
- Principle: Pyrosequencing measures the release of pyrophosphate during DNA synthesis, which is proportional to the amount of each nucleotide incorporated. This allows for the quantification of methylation levels at specific sites.
- Advantages: High sensitivity and accuracy, can quantify methylation levels.
- Disadvantages: Limited throughput, can be expensive.
4. Microarrays
- Principle: Microarrays use probes that bind to specific DNA sequences, including those with methylated cytosines. The intensity of the signal indicates the level of methylation at each site.
- Advantages: High throughput, can analyze methylation levels at thousands of sites simultaneously.
- Disadvantages: Limited resolution, may not be as accurate as other methods.
5. Next-Generation Sequencing (NGS)
- Principle: NGS allows for the sequencing of millions of DNA fragments simultaneously, providing high-resolution methylation data across the entire genome.
- Advantages: High throughput, comprehensive methylation profiling, can identify novel methylation patterns.
- Disadvantages: Expensive, requires specialized equipment and expertise.
These are just a few examples of the many methods available for checking methylation levels. The choice of method depends on the specific research question, budget, and available resources.
