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The use of magnetic particles for DNA isolation from selected spices
Gaňová, Martina ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of DNA from plant tissue of the required quality is very complicated, especially because of the presence of substances that can interfere during amplification of DNA. These substances are mainly polyphenols, polysaccharides, proteins and various dyes. The chemical diversity of such materials can have a significant effect on the yield and quality of DNA using one isolation procedure. The main aim of the work was to evaluate the use of microisolation protocol for related matrices to the quality of the isolated DNA as well as the evaluation of the effect of inhibitors isolated with the nucleic acid to the amplification in the PCR. DNA was isolated from dried paprika (Capsicum annuum). In the first step, the samples were homogenized using a lysis reagent with cetyltrimethylammonium bromide. Subsequently, the DNA was purified by reversible adsorption on magnetic particles. It was tested six different modified particles. The concentration and purity of the obtained DNA was determined by spectrophotometry measuring the absorbance of the DNA solution in TE buffer. The quality of the DNA was confirmed by amplification in PCR. For the PCR were used primers specific for plant ribosomal DNA (rDNA). The presence of PCR products was detected by agarose gel electrophoresis. It was found out that used microassay is suitable for isolating of the DNA of the corresponding purity that is suitable for the genetic analysis by PCR. The differences were found between the magnetic particles that were tested for DNA isolation.
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The influence of RDR6 activity and mode of RNAi induction on dynamics and mechanism of silencing of the reporter GFP gene in tobacco cell line BY-2
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee)
RNA interference (RNAi) is a process mediated by small RNAs (sRNA), which is significantly involved in the regulation of gene expression in plants. Diverse RNAi pathways can be divided into two basic mechanisms, which are post-transcriptional and transcriptional gene silencing (PTGS and TGS). Production of sRNAs is dependent on the presence of a double-stranded RNA molecule (dsRNA), which is cleaved by one of DCL proteins to produce sRNAs usually of 21-24 nt in length. One strand of the sRNA is subsequently loaded onto AGO protein. During PTGS, the AGO-sRNA complex interacts with the target RNA based on its sequence complementarity to the sRNA and cleaves it or blocks its translation. In the case of TGS, AGO interacts with plant-specific RNA Pol V and its transcripts, which are again complementary to the sRNA. This interaction allows assembling of a protein complex facilitating DNA and histone methylation inhibiting RNA Pol II transcription. There are numerous ways the dsRNA can arise. A significant part of dsRNA cell production is dependent on synthesising the complementary strand of the dsRNA by RDR6 (RNA-dependent RNA polymerase 6). RDR6 is also involved in the process of the secondary sRNA formation. The significance of RDR6 during PTGS was examined using a GFP reporter gene either during...
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Study of RNAi mechanisms in tobacco BY-2 cell line and potato plants
Tyč, Dimitrij ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee) ; Moravec, Tomáš (referee)
Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...
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Isolation and detection of DNA from plant species important for food prodution
Orel, Matúš ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In the food industry, it is very important to take care of the quality, safety and organoleptic properties of the products supplied. For this reason, food must be checked. However, not all information can be found using conventional techniques such as immunoassays, chromatographic techniques, etc. DNA-based techniques can be used for these cases where traditional procedures are insufficient. Among them, the best known technique is PCR. The aim of the thesis was to isolate DNA from vegetable samples (broccoli, beetroot, carrot and pepper). DNA was isolated using the magnetic particle method and the traditional CTAB method. Both methods were able to isolate the DNA from the vegetable samples in quality and at a concentration suitable for PCR, where the 35S rDNA gene region was amplified (more precisely about 700 bp of the 18S-ITS1-5,8S region). After amplification, the PCR products were subjected to restriction reactions and the results compared to bioinformatic analysis. These steps have succeeded in finding suitable enzymes for diferentiation of PCR products from the tested vegetable species.
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Evaluation of a micromethod for isolation of DNA from plant leaf, fruit and fruit products
Balažovičová, Nikola ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The thesis has been focused on testing of micromethod of DNA isolation from leaves, fruits and fruit products. Jams were selected for the analysis of plant DNA in technologically processed foods. Plant leaves, fruits, and jams were homogenized using plastic copist in a lysis buffer containing 2% cetyltrimethylammonium bromide (CTAB) with 2.5M sodium chloride (NaCl). Microisolation of plant DNA was performed using poly(hydroxyethylmethacrylate-co-glycidylmethacrylate) – P(HEMA-co-GMA)microparticles. Isolated the DNA concentration and purity were assessed by UV light aborbance using a spectrophotometer. After that, amplification of the DNA was tested in PCR. Primers specific for plant ribosomal DNA: 18S_for a 5,8S_rev (PCR product - 700bp), 26S_for a 26S_rev (PCR product - 220 bp), 18S_for a 18¬S_rev (PCR product - 263 bp) were used. The PCR conditions were optimized and the effect of the amplicon length on its detection was followed. PCR products were detected by agarose gel electrophoresis. It was shown that DNA isolated from almost all of leaves using magnetic particles was in PCR-ready quality in contrary to the fruits. DNA amplified in PCR with primers giving short PCR products was isolated from almost all tested jams. The method must be optimalised, yet.
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The role of hybridization in plant evolution - using different methods for detecting plants of hybrid origin in the Elytrigia repens - Elytrigia intermedia hybrid complex
Paštová, Ladislava ; Krahulec, František (advisor) ; Kovařík, Aleš (referee) ; Blattner, Frank (referee)
Hybridization is an important phenomenon in plant evolution because it is one of the sources of new genetic variability. Hybridization is the merging of genomes of formerly isolated evolutionary lineages. In many taxonomic groups, the detection of plants of hybrid origin is challenging. A wide spectrum of methods for their detection has been employed since the beginning of botanical research. The introduction of genomic in situ hybridization has had a great impact on the study plants of hybrid origin. This molecular cytogenetic approach allows to reveal the genomic contributions of particular parental species to hybrid taxa. The tribe Triticeae is a prime example of a group whose present-day diversity has been strongly influenced by hybridization (together with polyploidy). The majority of its species are allopolyploids resulting from frequent interspecific and intergeneric hybridization. The structure of relationships within the tribe is therefore highly reticulate. This thesis includes three papers dealing with the hybrid complex of Elytrigia repens - E. ×mucronata - E. intermedia: (1) The representatives of this hybrid complex are morphologically poorly differentiated, and only two morphological characters are used to their distinguishing. Among anatomical characters on the leaf blade, some...
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Comparison of different types of magnetic carriers for DNA microisolation from foods
Koplík, Jerguš ; Lunerová,, Jana (referee) ; Kovařík, Aleš (advisor)
Micro-isolation of PCR-ready from fresh and dried legumes seeds and food products containing legumes (hummus) was tested. For optimization process magnetic microparticles PGMAox was used. Optimum weight plants and food material was 200 mg. For isolation of DNA from fresh legumes, mixture containing 500 L of CTAB extraction buffer, 1 L -mercaptoethanol and 500 L chloroform-octanol was used. For isolation of DNA from dried legumes seeds and food products volume of CTAB extraction buffer was increased to 1 000 L. To achieve higher purity of DNA some prepared homogenates was precipitate by isopropylalcohol. The optimized process of DNA isolation was used to prepare a homogenate from food products which DNA was isolated by different types of magnetic carriers. For comparison, non-porous magnetic carriers poly(glycidyl methacrylate) PGMAox, poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) P(HEMA-co-GMA)ox covered by carboxyl groups and porous fully crosslinked microparticles poly(styrene-co-divinylbenzene) HPS-B-M-22-NH2, magnetic porous glass MPG and nanoparticles of iron oxides covered by poly(L-lysine) PLL were used. In average the highest concentration and the best purity of DNA was isolated by magnetic carriers P(HEMA-co-GMA)ox a PGMAox.
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Sample preparation for DNA analysis from foods of plant origin
Silná, Renata ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of high quality DNA is nessecary for many molecular biology applications. However, plant DNA contains high amonts of polysaccharides, polyphenols and various secondary metabolites, which decrease yield and quality of isolated DNA. The aim of this study was preparation of samples and different food matrices for DNA isolation DNA by magnetic particles. It was about 5 species of vegetable and 10 species of processed plant food. Homogenization of samples was performed in CTAB buffer. Isolation of plant DNA was performed by magnetic particles covered with carboxyl groups. All DNAs were isolated in conventional PCR qualities using primers for 700 bp amplicons, in the case of heat processed products for 220 bp ampilicons and for real time PCR. The efficiancy of separation of magnetic particles with DNA by magnetic separator and magnetic needle was compared. It was find out that DNA of higher purity was isolated using magnetic needle. The micromethod of isolation of plant DNA from homogenates with CTAB with magnetic particles is suitable for different processed food.
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The use of magnetic particles for isolation and purification of DNA from products for children nutrition
Pešková, Aneta ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
Isolation of plant DNA is complicated due to the presence of polyphenols, polysaccharides and other metabolites, that are isolated together with DNA. These compounds can affect the yield and quality of DNA and can inhibit a polymerase chain reaction (PCR). A modern, simple, and fast method of DNA isolation in molecular biology laboratories is magnetic separation based on reversible DNA immobilization on magnetic particles. These methods allow to obtain DNA of high quality and purity. In the experimental part, magnetic microparticles PGMA 2 mg/ml and magnetic nanoparticles functionalized by polylysine (PLL) 0,2 mg/ml were used for isolation of plant DNA from vegetables (carrots), fruits (pear, apple, lemon, mango) and heat treated food products for children (Hami first carrot, Nestlé fruit pocket, dmBIO pear carrot apple, Hello fruit snack with apples and Hello fruity snack with mango). The efficiency of separation of magnetic particles with bound DNA using a magnetic needle and a magnetic separator were compared. The quality and quantity of isolated DNA were verified by spectrophotometric analysis. The amplificability of isolated DNA was tested in a conventional PCR using primers specific for plant ribosomal DNA (rDNA). PCR products were analyzed by agarose gel electrophoresis. Major fluorescent bands were of 700, 350 and 220 bp corresponding to three different rDNA amplicons. DNA was isolated from heat treated food products for children in a PCR-ready quality. Only 220 bp long PCR products were detected, that indicated DNA degradation. The identity of PCR products was determined by restriction fragment lenght analysis (RFLP) using NlaIII enzyme cutting the rDNA subregion (ITS1) of Daucus carota (carrot). The digestion profiles were in a good agreement with those predicted from bioinformatic analysis confirming, thus, the specificity of the developed PCR method.
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DNA isolation from selected vegetable products (paprika)
Gőghová, Sabína ; Kuderová,, Alena (referee) ; Kovařík, Aleš (advisor)
The diploma thesis deals with micromethod of DNA isolation from ten differently processing food products containing pepper (Capsicum annum). PCR ready DNA was isolated by magnetic particles PGMA functionalized by carboxyl groups from homogenates prepared in lysis buffer with CTAB. Quantity and quality of DNA was estimated using spectrophotometric measurements and verified using PCR methods with primers specific for plant rDNA. Quality of isolated DNA varied depending on processing technology. DNA isolated from smoked grinded peppers and from heat treated food products was degraded and amplified with primers F_26S and R_26S (PCR product 220 bp) in contrary to the primers F_18S and R_5.8S (PCR product 700 bp). DNA isolated from the other food products was amplified with primers F_18S and R_5.8S (PCR product 700 bp). PCR product from one grinded pepper (Žitavská paprika) was cloned and sequenced.
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