The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research

Abstract

Methylation at position 5 of cytosine in DNA is an important event in epigenetic changes of cells, the methylation being linked to the control of gene functions. The DNA methylation can be analyzed by bisulfite genomic sequencing, and a large body of data have now been accumulated, based on which causation of diseases, for example cancer, and many other manifestations of cellular activities have been discussed intensively. This article gives an extensive account of the chemical aspects of bisulfite modification of cytosine and 5-methylcytosine in DNA. Various factors that affect the action of bisulfite are discussed, and a recent progress from our laboratory is explained. Conventional procedures for the bisulfite treatment consist incubation of single-stranded DNA with sodium bisulfite under acidic conditions. This treatment converts cytosine into uracil, but 5-methylcytosine remains unchanged. Amplification by polymerase chain reaction (PCR) of the bisulfite-treated DNA followed by sequencing can result in revealing the positions of 5-methylcytosine in the gene. We have discovered that the whole procedure can be significantly speeded up by the use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. Another recent finding is that urea, which has been often added to the reaction mixture with the purpose of facilitating the bisulfite-mediated deamination of cytosine in DNA, may not work as anticipated: we have observed that urea does not show such promoting actions in our treatments of DNA. A laboratory protocol for quantifying bisulfite, suitably simple for routine practice to ensure valid experiments, is described.