Chapter 25, Problem LGP

Research suggests that expression of certain genes is controlled by conversion of some cytosine bases in the genome to 5-methylcytosine by an enzyme called DNA methyltransferase. Cytosine methylation may be a means by which some genes are turned off as cells differentiate during growth and development. It may also play a role in some cancer processes and in defending the genome from foreign DNA such as viral genes. Measuring the level of methylation in DNA is an important analytical process. One method for measuring cytosine methylation is known as methylation-specific PCR. This technique requires that all unmethylated cytosines in a sample of DNA be converted to uracil by deamination of the C4 amino group in the unmethylated cytosines. This is accomplished by treating the DNA with sodium bisulfite $\left(NaHS{O}_3\right)$ to form a bisulfite addition product with its unmethylated cytosine residues. The cytosine sulfonates that result are then subjected to hydrolysis conditions that convert the C4 amino group to a carbonyl group, resulting in uracil sulfonate. Finally, treatment with base causes elimination of the sulfonate group to produce uracil. The modified DNA is then amplified by PCR using primers designed to distinguish DNA with methylated cytosine from cytosine-to-uracil converted bases. Write detailed mechanisms for the reactions used to convert cytosine to uracil by the above sequence of steps.