Supervisor: TÜ MRI professor Maido Remm.
Opponent: Teadur Aleksander Pozhitkov, Periodontoloogia osakond, Washingtoni Ülikool, Seattle, Ameerika Ühendriigid.
Nucleic acids are unique among all organic macromolecules by the ability to encode, decode and transmit digital information. This property is used in emergent technologies as diverse as medical diagnosis, nanoscale engineering and information storage. Still it is important to understand that the basis of this digital information processing are the chemical properties of nucleic acids, the most important being the spontaneous formation of double-stranded helix between complementary or semi-complementary single-stranded molecules, called hybridization. Taking into account the thermodynamic properties of nucleic acid hybridization allows researchers to model the process with great accuracy and thus improve many associated technologies. In current thesis the hybridization model is used to optimize multiplex PCR and microarray hybridization. We developed an efficient algorithm to distribute PCR primer pairs into multiplex groups based on their compatibility with each other. The algorithm is also implemented as both standalone and web-based computer program. We analyzed the probable causes of failure of multiplex PCR and demonstrated that the large number of nonspecific hybridization sites in template DNA is detrimental to PCR quality. Primer pairs with too many nonspecific hybridization sites not only worked poorly but caused the failure of other primer pairs as well. We developed a computer program to generate exhaustive list of all possible hybridization probes for the detection of bacterial tmRNA, capable of distinguishing between two groups of source RNA. The probes were evaluated on microarray and shown that by keeping the hybridization energy cutoff between target and non-target groups over 4 kcal/mol all false-positive signals were eliminated. We analyzed the possibility of increasing the hybridization speed of bacterial tmRNA on low temperatures by applying short specific oligonucleotides that selectively hybridize with template molecules and break their secondary structure. Using this method the hybridization speed was increased fourfold at 37C.