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DNA Extraction of Sweet Cherry (Prunus avium L.)
Hřebcová, Kateřina ; Sedlák, Petr (advisor) ; Korecký, Jiří (referee)
Isolation of high quality DNA in satisfactory yield and purity is a fundamental and essential step for all molecular-biological studies and analyses. The process of its extraction can be complicated by many of materials like are polyphenols, polysaccharides, proteins and other metabolites that can be co-isolated with nucleic acids and can act as inhibitors of PCR and cause deterioration of samples for further analyses.
In this thesis, mostly used methods of plant DNA isolation were mapped, and, in experimental part, results, regarded to the yield and purity, of selected plant DNA isolation methods were compared. DNA was obtained from various tissues of Prunus avium L. species, namely from fresh leaves, buds and from frozen embryos of several varieties. Comparison of the two commercial isolation kits (DNeasy Plant Mini Kit by Qiagen and GeneEluteTM Plant Genomic DNA Miniprep Kit, Sigma Aldrich) was the original intention. The first of the kits was replaced by simple and quick DEP-25 DNA Extraction Kit, Top-Bio and the experiment was extended with CTAB DNA isolation protocol, both with and without application of RNase into the protocol.
The results obtained proved quite significant differences between the methods used, both in yield and purity. The original assumption, supported by several studies, that commercial kits not always gain relevant results, regarded to ability to provide pure DNA, was not accurately proven, the assumption that the CTAB protocol can gain satisfactory results according to the DNA yield and purity was proved only with some tissues. The results of the spectrophotometry were supported with polymerase chain reaction (PCR) analyses conducted with the isolated DNA samples and after statistical evaluation were discussed.
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Analysis and characterization of BRCA1 splicing variants.
Hojný, Jan ; Kleibl, Zdeněk (advisor) ; Souček, Pavel (referee)
The Breast cancer gene 1 (BRCA1) codes for nuclear phosphoprotein with a key function in the regulation of DNA damage response. The BRCA1 protein contributes to the formation and regulation of protein supercomplexes that participates on the DNA double-strand break repair. These protein supercomplexes are formed by the protein-protein interactions between highly conservative protein motives in BRCA1 and its binding partners. Except to the wild type form of BRCA1 mRNA containing entire set of 22 exons coding for the 220 kD protein, numerous alternative splicing variants (ASVs) BRCA1 mRNA has been described. These ASVs code for BRCA1 isoforms lacking several critical functional domains. It has been proposed, that formation of BRCA1's ASVs represent a tool for regulation of BRCA1 function. Only poorly has been characterized a complex catalogue of in various human tissues and their expression. This study aims to address these questions. We optimized the identification of BRCA1's ASVs including those covering the entire transcripts of the wt BRCA1 mRNA with length exceeding 5.5 kb. In further analysis, we characterized 13 BRCA1's ASVs in RNA samples isolated from peripheral blood mononuclear cells (PBMNC) obtained from patients with breast cancer (BC) and control subjects. The majority of the identified...
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Antioxidants in human nutrition
Nemcová, Romana ; Hučko, Boris (advisor) ; Hroncová, Zuzana (referee)
The aim of this study was to show the importance of the antioxidants in human nutrition, their functioning and effects on human health. Special attention is recently paid to antioxidants due to their positive effects on the human body. Antioxidant activity is variable which evaluates the ability of the body to capture the free radicals, to prevent their occurrence or to convert them to unreactive or less reactive forms. Free radicals are formed in the human body as a byproduct of metabolism in the cells, where they play a positive role in immunity. On the other hand, their excessive production may be detrimental to the organism, they disrupt cell membranes, destroy DNA. Excessive formation of reactive oxygen and nitrogen free radicals arises oxidative stress. Oxidative stress significantly contributes to the development of various degenerative diseases and tumors, it causes DNA mutation and it damages macromolecules and tissues.
Human organism has developed its own antioxidant activity in the form of endogenous antioxidants which are produced by the body. Exogenous antioxidants are received from food and they are mainly of plant origin. The antioxidant enzymes (SOD, catalase, peroxidase), vitamins, carotenoids and phenolic compounds are the most important. Antioxidants perform important functions in the food industry, in which they prevent oxidation of fats and proteins, maintain the taste and colour of the food and also increase their durability.
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Analýza polymorfizmu DNA pšenice
Pospiš, Matěj
Common wheat is the most important and widespread agriculture plant. It is used mainly in bakery industry, or as fodder and it is the major human nutrition source. Its genotypes cause different kinds of wheat colouring: blue, purple, yellow or white. This colouring is caused by anthocyanins with antioxidant effects. The positive influence of these antioxidants on human are considered to be healthy. Summer and winter wheat samples with white caryopsis (Novosibirskaja 67 and Heroldo) and blue aleurone (UC66049, Tschermaks Blaukörniger Sommerweizen, Tschermaks Blaukörniger, 48M, Skorpion, RU 440-6, RU 440-5, Barevna 9, Barevna 25, Xiao Yian, EF 02-54/9 and H 90-15-2) were chosen for the analysis. These samples were taken from Agrofest fyto Kroměříž s.r.o collection. DNA was isolated after these samples germination and the analysis was done on the basic of DNA polymorphism based on PCR reaction. There were three observed features: Locuses Glu-A3 for gluten low-molecular subunits which influence baking quality. The second observed feature was the allele's identification that is responsible for puroindoline (Pina and Pinb) genes production and these genes are responsible for the caryopsis hardness. The very last of observed features were Waxy genes and detection of Waxy genes null allele. The expression of these null allele influences the starch quality in caryopsis. Final outcomes can be used in wheat breeding.
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Properties and function of middle T antigen of the murine polyomavirus
Fabiánová, Anna ; Forstová, Jitka (advisor) ; Čáp, Michal (referee)
Polyomaviruses are small DNA viruses, which are able to induce a broad variety of tumors. The main oncoprotein of the mouse polyomavirus (MPyV) is middle T antigen (MT antigen) which is able to transform cells. MT antigen has not an enzymatic activity of its own. It is able to activate signal transduction of host cells through its interactions with certain cellular proteins. These proteins include protein phosphatase 2A (PP2A), Src kinase, phosphatidylinositol 3 kinase (PI3K), Shc protein, 14-3-3 protein and phospholipase Cγ1 (PLCγ1). This work is focused on interaction between MT antigen and cellular proteins and on the impact of this interaction on cell transformation. Since MT antigen is a potent oncogene, the work also deals with the character of transformed cells and tumor development in mouse mammary epithelium. Keywords: polyomaviruses, MT antigen, PP2A, PI3K, PLCγ1, Shc protein, 14-3-3 protein
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Microsatellite instability in hereditary nonpolyposis colorectal cancer patients
Sekowská, Martina ; Křepelová, Anna (advisor) ; Mareš, Jaroslav (referee) ; Kleibl, Zdeněk (referee)
Detection of microsatellite instability (MSI) is the standard part of mutational analysis in hereditary nonpolyposis colorectal cancers (HNPCC). Characteristic phenotypic feature of MSI indicates loss of mismatch repair (MMR) in tumor cells. We studied MSI in 205 tumors from 152 patients with HNPCC. Of these, 37 patients fulfilled Amsterdam criteria, 72 patients were familial and 43 were sporadic cases. We used methods of fragmentation analysis on polyacrylamide gel and/or with fluorescent labelled primers (ABI Prism 310 Genetic Analyzer). Three mononucleotide (BAT-RII, BAT-25, BAT-26) and five dinucleotide (D2S123, D3S1029, D5S346, D17S250, D18S58) repeat loci were analysed. We detected 75 tumors with high degree of MSI (MSI-H), 12 tumors with low degree of MSI (MSI-L) and 118 tumors with stable microsatellites (MSS). In 44 of these, loss of heterozygozity (LOH) was found. In 30 patients with MSI-H tumors a mutation in one of mismatch repair genes was detected. Microsatellite analysis was positively correlated with immunohistochemical detection of MLH1 and MSH2 proteins.
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PCR based assay for detection garden pea lec gene
Vráblík, Aleš ; Hodek, Jan ; Ovesná, Jaroslava
The method was developed and verified by staff of National reference laboratory for GMO identification and DNA fingerprinting suited for governmental control laboratories and private laboratories that analyze food and feed derived from various plant matrices. Method can be applied for quantification of GM pea in a sample, when reference gene is used as a standard. The method describe internal garden pea specific gene, lektin in this case, detection using PCR and real-time PCR. The general principle of the assay relies on lektin specific sequence presence that represents a species specific gene of garden pea. In many matrices DNA is damaged so amplification of short stretches of DNA is used. After amplification of target exploiting newly developer species specific gene primers PCR products are separated by electrophoresis in agarose gel. Resulting band is visualized by UV light and its position is compare with size standard. Alternatively, real-time PCR and ABI platform in combination with SYBR® Green can be used. Reaction parameters are described and specificity of reaction was verified. LOD as well as LOQ was defined.
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Application of DNA barcoding on genus \kur{Folsomia} (Collembola) and mitochondrial geonome of \kur{F. candida}
SLÁMOVÁ, Martina
Mitochondrial molecular marker COI was tested for use in species identification of selected species of genus Folsomia (Collembola). Marker was succesfuly amplified and sequenced. Dendrogram constructed by Neighbor-Joining method with Kimura-2-Parameter model grouped all individuals into presumed species clusters and high intraspecific variability of F. quadrioculata suggests the existence of cryptic species. Furthermore, 65 % of mitochondrial geonome of F. candida was obtained with 16 tRNA genes, 9 proteincoding genes and 2 rRNA genes identified. So far the genome characteristics correspond to the one described in G. hodgsoni.
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