Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing.

Here we report on the modification of a genomic sequencing protocol, published by Frommer el al. (6), which allows the determination of the methylation status of cytosine residues on individual chromosomes. The method is based on PCR amplification from chemically modified genomic DNA, in which unmethylated cytosine residues have been converted into uracil by deamination. Sequencing of individual PCR products establishes the position of each 5-methylcytosine on individual chromosomes (6). On single-stranded DNA, a high concentration of sodium bisulphite at pH 5.0 induces deamination of cytosine residues but not of 5-methylcytosine residues (1—4). In double-stranded DNA, the rate of cytosine deamination is less than 0.1 % of the rate in single-stranded DNA (4,5). We have applied bisulphite genomic sequencing to study DNA methylation in the mouse Insulin-like growth factor 2 (Igfl) gene (7). Initially, we chose the published reaction conditions (6), but used glass beads for the subsequent purification of the DNA. On several independent reactions, not all the unmethylated cytosine residues had become deaminated. In the sequenced PCR products clustered stretches of DNA remained unmodified, presumably because they had become double-stranded during treatment (Fig. 1 A). We therefore introduced the following modifications in the protocol which resulted in full chemical conversion in most of the PCR products analysed (7, Fig. IB): 1, DNA was alkalinedenatured directly prior to treatment; 2, the DNA concentration was decreased and the bisulphite concentration increased and 3, treatment was performed at a lower temperature to increase the extent of cytosine sulfonation at pH 5.0 (5) and to reduce annealing of single-stranded DNA sequences during treatment. These modifications did not affect 5-methylcytosine residues which remained unconverted (7).