| Abstract [eng] |
Synthetic base-modified nucleic acids are widely used in various fields. The aim of this dissertation was was to investigate the properties and utility of oligonucleotide-tethered 2′,3′-dideoxyribonucleoside 5′-triphosphates (OTDDNs), with emphasis on labeling of DNA and cDNA molecules for high-throughput sequencing applications. It was demonstrated that OTDDNs are substrates for a range of DNA polymerases of families A, B, X and RT. By means of in vitro evolution we have selected T7 RNA polymerase mutant with relaxed substrate discrimination that is able to synthesized chimeric nucleic acids labeled by OTDDNs. Moreover, DNA polymerases that perform synthesis through the unnatural linkage within OTDDN were identified. Several known NGS library preparation methods rely on chemoenzymatic approaches, however such techniques suffer from poor efficiency and laborious protocols. In this work modified terminators enabled the integration of fragmentation and sequencing adapter addition into a single enzymatic step. This in turn opened doors for the development of new DNA and RNA sequencing methods which have rapid and simple protocols and generate high-quality data. Two new OTDDN-based methods described in the dissertation include novel microbiome analysis technique st16S-seq and novel gene expression analysis method MTAS-seq. Taken together, this work suggests OTDDNs as a promising tool for high-throughput nucleic acid analysis. |
