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Development of the new method for in vitro selection of DNA aptamers
Bláhová, Kamila ; Míšek, Jiří (advisor) ; Moserová, Michaela (referee)
Aptamers are short sequences of single-stranded DNA or RNA that are able to specifically bind various molecules (drugs, lipids, sugars, proteins, etc.). These aptamers are isolated from large libraries of random oligonucleotide sequences by SELEX (Systematic Evolution of Ligands by Component Enrichment) or in vitro selection. Despite the success of this method, in vitro selection often requires more than ten rounds of affinity selection as well as optimization of selection conditions. To increase the efficiency of aptamer selection, several methods have been developed that use an increase in the number of secondary structures in random oligonucleotide libraries. These methods, based on increasing the possibility of canonical base pairing in single-stranded oligonucleotides, increased the efficiency of the in vitro selection method. In this work, it was tested whether increasing the probability of occurrence of G-quadruplexes, as structural motifs in random sequences, will lead to increased efficiency of aptamer selection. Four single-stranded DNA libraries with different numbers of guanine (25 %, 35 %, 45 %, 55 %) in a random sequence were used. Streptavidin was chosen as the model molecule for selection, against which several aptamers that are not rich in guanine (G) have previously been selected....
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Detekce fytoplazmy Candidatus Phytoplasma mali metodou dřevinných indikátorů
Blahová, Kamila
A subject of the diploma thesis was the optimization of a procedure of detecting a phytoplasma Candidatus Phytoplasma mali using woody indicator Golden Delicious in greenhouse conditions as an alternative method to the phytoplasma presence testing by biological indexing in field conditions. To control the presence of phytoplasma in the experimental plants, molecular method of real-time PCR was used. The experiment was set up on the demonstration area of the Department of Breeding and Propagation of Horticultural Plants in the grounds of Mendel University in Brno. Three different ways of transferring of the phytoplasma infection were compared: method of parallel grafting of the tested variety and of the indicator on the seedling in the period of dormancy (method number 1), method of summer budding of the tested variety on indicator grafted on the seedling in the period of dormancy (method number 2), and method of double summer budding of the indicator and of the tested variety on the seedling (method number 3). On a woody indicator Golden Delicious, varieties Virgin Czech, Canadian rennet, Gascoigne, Ribston and Boskoop were tested, 10 plants of each variety. The experiment lasted 10 months. A total of 150 tested plants were assessed by a visual inspection of the symptoms presence. During the monitoring period, none of the tested plants showed typical symptoms of the apple proliferation. Presence of the Candidatus Phytoplasma mali was verified using real-time PCR method in 60 tested (indicator) plants. The infection was confirmed in 12 of the indicator plants. The detected quantities of the phytoplasma in the tissues however reached very low values. The highest number of the infected plants was found in tests of the Gascoigne variety, established by the method of double summer budding of the indicator and of the tested variety on the seedling (method number 3). This method provided the most successful inoculation of the indicator by the phytoplasma. Based on the results obtained during the presented study, it can be declared that biological testing of the phytoplasma presence using short-term greenhouse tests is not reliable. When using testing with the indicator ´Golden Delicious´, it is recommended to realize the experiments for at least two growing seasons, as it is in the field tests. Due to the fact that during the time-limited realization period of the experiment in this study it was found that the phytoplasma was not sufficiently propagated, the final results of the comparison of the indicator plants inoculation methods will be available at the end of autumn of 2017.
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Proliferace jabloně (Candidatus Phytoplasma mali)
Blahová, Kamila
The aim of this thesis was to write a literature review on the topic of apple proliferation. To achieve this goal, a basic overview of the history of phytoplasma and determining their terminology were created. Taxonomy of Candidatus Phytoplasma mali phytoplasma, its bionomics, host plant species, geographic distribution and economic importance were mentioned in this work. Moreover symptoms on infected apple trees, the factors that influence the spread of the disease and its subsequent detection of available diagnostic methods as well as. Possible ways of transmission, bionomics of insect vectors, safeguards and regulatory standards were deseribed. In the part related to one's own comments, history of Ca. P. mali presence, the apple production orchards area and the current situation of phytoplasma expansion in the Czech Republic together with the recommendation of prevention against further spread of the pathogen are presented.
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