DNA methylation: Bisulphite modification and analysis

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Abstract

DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions—in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.

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Figure 1: Schematic diagram of the five critical steps in the bisulphite conversion and PCR amplification reaction.
Figure 2: Comparison of efficiency of bisulphite PCR amplification after different desalting and desulphonation procedures.
Figure 3: Examples of bisulphite PCR amplification evaluation.
Figure 4: Typical examples of direct and clonal PCR sequencing analysis after bisulphite conversion.

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Acknowledgements

The authors thank Rebecca Hinshelwood for help with the figures. The work was partly supported by grants from NH and MRC to S.J.C. (ID9293811, ID293810, ID325622).

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Correspondence to Susan J Clark.

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The authors declare no competing financial interests.

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