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Cell Biology JoVE (Open Access) Citable · DOI

DNA Methylation: Bisulphite Modification and Analysis

DOI: 10.3791/3170-v
What you'll learn
  • ✓ Perform bisulphite conversion to deaminate unmethylated cytosines
  • ✓ Distinguish methylated from unmethylated DNA cytosines via PCR
  • ✓ Analyze DNA methylation patterns using genomic sequencing
  • ✓ Interpret methylation analysis results from bisulphite-treated samples
Protocol

Biopharma Insights The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Difficulty
intermediate
Total time
~4–6 hours per sample (including bisulphite treatment, PCR, and sequencing)
Biosafety
BSL-1

Steps

1
Prepare and denature genomic DNA

Extract and prepare genomic DNA, then denature it to single-stranded form to expose cytosine residues for bisulphite modification.

▶ 01:52
2
Perform bisulphite deamination reaction

Incubate denatured DNA under acidic bisulphite conditions to convert unmethylated cytosines to uracil while leaving 5-methylcytosines unchanged.

▶ 02:57
3
Amplify bisulphite-converted DNA by PCR

Use PCR to amplify the target genomic region from bisulphite-treated DNA, converting uracil to thymine and enabling distinction of methylation status.

▶ 06:38
4
Analyze and interpret methylation patterns

Sequence PCR products and analyze the distribution of converted versus unconverted cytosines to determine DNA methylation status at target sites.

▶ 09:08
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