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Biochemical analyses of the DNA interaction partners
Valchová, Michaela ; Coufal,, Jan (referee) ; Brázda, Václav (advisor)
This thesis was focused on DNA analysis. The fluorescently labelled oligonucleotides were at first hybridised and subsequently analysed by HRM analysis to determine the melting temperatures of the oligonucleotides depending on the environment. This thesis describes the change of melting temperature of oligonucleotides in environments containing mono and bivalent ions and the influence of protein binding on the stability of these DNA structures. From determined melting points, it was specified whether the ion/protein stabilised or destabilised the oligonucleotide. Furthermore, plasmids were isolated and analyzed by atomic force microscopy.
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Analysis of the composition of selected probiotic products by PCR-HRM
Tomanová, Barbora ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
This work was focused on the detection of probiotic bacteria in four different probiotic products (probiotic cream, probiotic tampons, oral probiotics and soy beverages with probiotics). The viability of the bacteria contained in the products was verified. Complex matrices of the products were used to isolate DNA in a quality suitable for the PCR method, followed by identification of the declared bacterial genus and species. Amplification was achieved with conventional PCR and real-time PCR, genus- and species-specific primers were used. Bacteria, of the genus Lactobacillus and Bacillus and bacterial species Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus gasseri, were proven to be within the products. Subsequently, the DNA from mixed bacterial species in the probiotic tampon were distinguished using PCR-HRM. Five sets of primers were used to test this. Two sets of primers (primers P1V1, P2V1 and V1F-HRM, V1R-HRM) were evaluated as the most suitable for resolution.
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Magteic particles and their applications in biotechnology
Knápková, Monika ; Konečná, Jana (referee) ; Trachtová, Štěpánka (advisor)
The thesis is focused on the magnetic particles which are used in several biotechnological applications. The theoretical part deals with the specific properties of these nanoparticles and materials of which the nanoparticles can be made. There are also mentioned some of the biotechnological applications of magnetic particles. During the experimental part, selected types of magnetic particles were used to isolate nucleic acid. The quality of the isolated DNA with respect to purity was evaluated using the polymerase chain reaction and its modifications. High resolution analysis (HRM analysis) was also used to verify the quality of the isolated DNA and to resolution Lactobacillus casei and Lactobacillus rhamnosus. DNA isolation using magnetic carriers was successful. Commercially available MPG microcarriers and magnetic microparticles Fkol 77ox were the most suitable. In terms of purity magnetic nanoparticles F79/L3-PLL were the most suitable for the DNA isolation. The resolution of bacterial strains of Lactobacillus casei and Lactobacillus rhamnosus was not successful.
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Application of the method PCR-HRM analysis to identify bacteria in foods and food supplements
Šurková, Alice ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of the thesis was focused on foods and food supplements containing microorganisms, especially bacteria. Furthermore, the thesis deals with methods for identification of the bacteria, primarily polymerase chain reaction (PCR). The thesis also includes real-time PCR and is specially focused on high resolution melting analysis (HRM). During the experimental part, the DNA sample was isolated from a chosen probiotics product using magnetic microparticles. The concentration of the DNA sample was determinate and DNA was subjected to PCR with subsequent detection PCR products by agarose gel electrophoresis. To the results specify HRM analysis was then performed.
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Identification of DNA from plant foods using molecular techniques
Papala, František ; Langová, Denisa (referee) ; Němcová, Andrea (advisor)
The aim of this bachelor thesis is the identification of plant DNA in a complex matrix using molecular techniques. The work first focuses on a search of literature sources concerned with molecular techniques of DNA identification. Next, it deals theoretically with methods of isolation and characterization of plant DNA. Specifically, we shall work with blueberry DNA. In the experimental part, DNA from four commercial products was isolated and subsequently subjected to PCR and HRM analysis.
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Use of advanced molecular biology techniques for detailed characterization of probiotic bacteria from a dietary supplement
Folwarczná, Tereza ; Trachtová, Štěpánka (referee) ; Smetana, Jan (advisor)
The aim of the diploma thesis was the identification of probiotic bacteria from a probiotic food supplement in the form of syrup. DNA was isolated from the complex matrix of the product in sufficient purity and quality suitable for the real-time PCR method followed by PCR-HRM. Specific primers for the genera Lactobacillus, Bifidobacterium and Streptococcus and at the species level for the species Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus thermophilus were used for amplification by real-time PCR and PCR-HRM. After optimizing the conditions for specific primers, all declared bacteria were detected in the product.
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Identification of DNA from plant foods using molecular techniques
Papala, František ; Langová, Denisa (referee) ; Němcová, Andrea (advisor)
The aim of this bachelor thesis is the identification of plant DNA in a complex matrix using molecular techniques. The work first focuses on a search of literature sources concerned with molecular techniques of DNA identification. Next, it deals theoretically with methods of isolation and characterization of plant DNA. Specifically, we shall work with blueberry DNA. In the experimental part, DNA from four commercial products was isolated and subsequently subjected to PCR and HRM analysis.
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Biochemical analyses of the DNA interaction partners
Valchová, Michaela ; Coufal,, Jan (referee) ; Brázda, Václav (advisor)
This thesis was focused on DNA analysis. The fluorescently labelled oligonucleotides were at first hybridised and subsequently analysed by HRM analysis to determine the melting temperatures of the oligonucleotides depending on the environment. This thesis describes the change of melting temperature of oligonucleotides in environments containing mono and bivalent ions and the influence of protein binding on the stability of these DNA structures. From determined melting points, it was specified whether the ion/protein stabilised or destabilised the oligonucleotide. Furthermore, plasmids were isolated and analyzed by atomic force microscopy.
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Magteic particles and their applications in biotechnology
Knápková, Monika ; Konečná, Jana (referee) ; Trachtová, Štěpánka (advisor)
The thesis is focused on the magnetic particles which are used in several biotechnological applications. The theoretical part deals with the specific properties of these nanoparticles and materials of which the nanoparticles can be made. There are also mentioned some of the biotechnological applications of magnetic particles. During the experimental part, selected types of magnetic particles were used to isolate nucleic acid. The quality of the isolated DNA with respect to purity was evaluated using the polymerase chain reaction and its modifications. High resolution analysis (HRM analysis) was also used to verify the quality of the isolated DNA and to resolution Lactobacillus casei and Lactobacillus rhamnosus. DNA isolation using magnetic carriers was successful. Commercially available MPG microcarriers and magnetic microparticles Fkol 77ox were the most suitable. In terms of purity magnetic nanoparticles F79/L3-PLL were the most suitable for the DNA isolation. The resolution of bacterial strains of Lactobacillus casei and Lactobacillus rhamnosus was not successful.
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