Bisulfite Sequencing - detect DNA Methylation

Epigenetics & phenotype variation

Two mice with identical DNA sequences can have different phenotypes (e.g., coat color). Genes determine phenotype, but variation can arise without DNA sequence changes. Epigenetics explains this: same DNA sequence, but different epigenetic patterns (e.g., methylation, histone modification). In epigenetics, the DNA sequence is unchanged, but chemical modifications (like methylation of cytosine or histone modifications) alter gene expression and phenotype.

DNA methylation & gene inactivation

DNA methylation is one of the most abundant epigenetic modifications. In most animals, it occurs on cytosine residues. When cytosines in a promoter region are methylated, gene transcription is typically inactivated. Thus, promoter methylation = gene silencing.

Bisulfite conversion: principle

Bisulfite sequencing detects DNA methylation. It exploits the chemical difference between methylated vs. unmethylated cytosines.

Unmethylated cytosines → converted to uracil by bisulfite treatment.

Methylated cytosines → remain unchanged.
After PCR, uracil (U) is read as thymine (T). So unmethylated C → T, while methylated C stays C.

Bisulfite conversion: experimental outcome

After bisulfite treatment + PCR:

C → T = originally unmethylated cytosine.

C remains C = originally methylated cytosine.
By comparing treated vs untreated DNA sequences, methylation sites can be mapped.

Application: cancer biology

Bisulfite sequencing is useful in analyzing genomes to identify methylation patterns. For example, it can reveal silenced tumor suppressor genes, which are candidates for tumor promotion when inactivated. Thus, bisulfite sequencing is a tool for studying DNA methylation in disease contexts, especially cancer.