Does urea promote the bisulfite-mediated deamination of cytosine in DNA? Investigation aiming at speeding-up the procedure for DNA methylation analysis

Methylation of cytosine in DNA at position 5 plays important roles in gene functions. Changes in the methylation status are linked to cancer. These studies have been developed on the basis of determining 5-methylcytosine residues [mC] in DNA. This analytical procedure uses the principle that bisulfite deaminates cytosine [C] but it deaminates mC only very slowly. Thus, 'bisulfite genomic sequencing' involves treatment of a given DNA sample with bisulfite followed by PCR amplification and sequencing, through which C residues in the original DNA are found as T and mC as C. In this procedure, a treatment with 3-5 M sodium bisulfite for 12-16 hr at 55 degrees C has been conventionally used. Recently, we were able to improve the efficiency of this procedure by introducing a highly concentrated (10 M) bisulfite solution. Aiming at further improvement of the procedure, we have now explored the effect of adding urea in this bisulfite treatment, as urea was reported to improve the deamination efficiency. Using 7.5 M ammonium bisulfite (pH 5.4) at 70 degrees C with or without the presence of 6 M urea, we performed deamination and sequencing of a DNA sample having known multiple CpG sites with mC. The deaminated DNAs were then subjected to PCR amplification followed by sequencing. In the 15 min-treated sample, the deamination extents were; C 96.5%, mC 1.1% for "bisulfite-only"; and C 90.3%, mC 1.4% for "bisulfite + urea". In the 30 min-treated sample, these values were; C 99.7%, mC 3.6% for "bisulfite only"; and C 99.7%, mC 2.1% for "bisulfite + urea". These results indicate that urea did not enhance the deamination efficiency. In the PCR, we did not observe significant improvements regarding the amounts of DNA necessary to obtain adequate amplification. Urea at 2 M, 4 M, and 8 M, showed no improvements. We conclude that urea gave no significant effect in the bisulfite genomic sequencing of the DNA used.