This thesis will review the chemical background of most commonly used sequencing technologies. Since the development of first sequencing methods in 1977, sequencing has become a routine tool in molecular biology and medical research. During past decade the field has evolved rapidly leading to million-fold reductions in the cost of sequencing. Although the 2',3'-dideoxy Sanger method is still widely used, new methods referred to as next-generation sequencing have taken over the market. Development of efficient sequencing technologies has relied on major chemical and technical advances. Over the years, the fundamentals of sequencing have not changed, but the marked increase in efficiency is reached through massive parallelization of individual polymerase catalyzed DNA extension reactions. This has been enabled by introduction of chemically modified nucleotides with cleavable terminator groups and fluorescent dyes attached to them. In addition, the massive parallelization relies on advances in silica surface chemistry that allow DNA fragments to be covalently bound to solid supports, forming the grounds of DNA microarray technology. New developments in sequencing aim for the detection of single DNA molecules in real time during DNA synthesis and replacing the optical detection methods with electronic methods that allow recording of the sequence data in digital format.
The experimental part of the thesis studies selective oxidation of 5-hydroxymethyl cytosine to 5-formyl cytosine. In addition to the four alternating bases, DNA in living cells undergoes covalent modifications. These are referred to as epigenetic changes and they are thought to have important biological roles. A class of epigenetic modification involves the methylation and hydroxymethylation of the 5-carbon of cytosine. The modified cytosine residues are not distinguished from cytosine in traditional polymerase based sequencing. However, treating DNA with bisulfite prior to sequencing leads to deamination of cytosine, but leaves the modified cytosine residues unchanged. It was recently suggested that the methylated and hydroxymethylated cytosine residues could be distinguished from each other by selectively oxidizing the 5-hydroxymethyl cytosine to 5-formyl cytosine prior to bisulfite treatment. 5-formyl cytosine undergoes deamination in bisulfite treatment, which allows distinguishing between the two. We investigated six potential oxidizing agents for oxidizing 5-methylcytosine. For testing the agents, we used 5-hydroxymethyl uracil, 5-hydroxymethyl cytosine as model molecules. In addition, we synthesized a protected 5-hydroxymethyl-2'-deoxycytidine that could further be used for synthesis of 5-hydroxymethyl-2'-deoxycytidine phosphoramidite that can be then used in oligonucleotide synthesis. The preliminary results suggest that four agents (KRuO4, (NH4)2Ce(NO3)6, TPAP, and BaMnO2) could potentially be used for selective oxidation of 5-hydroxymethylcytidine in DNA. However, the compatibility of these agents should be carefully tested in DNA oligonucleotides and genomic DNA, as well as with available sequencing technologies.
