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DNA extraction from plant tissues for polymerase chain reaction analysis
Trojánek, Zdeněk ; RNDr.Roman Matyášek, CSc. (referee) ; Rittich, Bohuslav (advisor)
Extraction of nucleic acids is an important step for all molecular biological studies. The process of isolation of plant DNA is complicated due to the presence of polyphenols, polysaccharides and other metabolites. They can be co-isolated with DNA and act as PCR inhibitors. The aim of this study was to compare CTAB extraction procedure, Qiagen DNA easy kit, direct homogenization, carboxyl-functionalised magnetic non-porous HEMA based microspheres and combination of the above mentioned methods for DNA isolation from different plants. The DNA was evaluated regarding concentration, purity and amplification in PCR. All methods provided DNA that could be used in downstream PCR applications. However, there were differences regarding yield, purity, labour intensiveness and cost. Combination of direct homogenization and magnetic microspheres coated by carboxyl groups was isolated DNA from various plants and plant foods in a quality suitable for convectional PCR, real time PCR and restriction analysis. This method is fast, simple and does not require work with harmful substances.
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Analysis of rRNA genes in variets Brassica napus
Dofková, Květoslava ; RNDr.Roman Matyášek, CSc. (referee) ; Kovařík,, Aleš (advisor)
Brassica napus (AACC, 2n = 38) is an allotetraploid species derived from the parentel diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). The aim of thesis was to carry out the genetic and epigenetic analysis of high-copy rRNA genes (or rDNA) in several varieties of hybrid species B. napus. The experiments involved determining the ratio of parental genes in hybrids, sequencing and methylation analysis of the promoter region of rDNA. Using Southern hybridization, it was revealed significant variability in the number of parental rDNA units between each variety. Data from sequence analysis were in good agreement with the results of Southern blot. Genetic recombination between parental rDNA units was revealed in one variety by DNA sequencing of promotor region. To study methylation, bisulfite sequencing was performed. It was found out that rDNA units of B. rapa origin have a higher value of methylation than units originated from B. oleracea.
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DNA extraction from plant tissues for polymerase chain reaction analysis
Trojánek, Zdeněk ; RNDr.Roman Matyášek, CSc. (referee) ; Rittich, Bohuslav (advisor)
Extraction of nucleic acids is an important step for all molecular biological studies. The process of isolation of plant DNA is complicated due to the presence of polyphenols, polysaccharides and other metabolites. They can be co-isolated with DNA and act as PCR inhibitors. The aim of this study was to compare CTAB extraction procedure, Qiagen DNA easy kit, direct homogenization, carboxyl-functionalised magnetic non-porous HEMA based microspheres and combination of the above mentioned methods for DNA isolation from different plants. The DNA was evaluated regarding concentration, purity and amplification in PCR. All methods provided DNA that could be used in downstream PCR applications. However, there were differences regarding yield, purity, labour intensiveness and cost. Combination of direct homogenization and magnetic microspheres coated by carboxyl groups was isolated DNA from various plants and plant foods in a quality suitable for convectional PCR, real time PCR and restriction analysis. This method is fast, simple and does not require work with harmful substances.
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Analysis of rRNA genes in variets Brassica napus
Dofková, Květoslava ; RNDr.Roman Matyášek, CSc. (referee) ; Kovařík,, Aleš (advisor)
Brassica napus (AACC, 2n = 38) is an allotetraploid species derived from the parentel diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). The aim of thesis was to carry out the genetic and epigenetic analysis of high-copy rRNA genes (or rDNA) in several varieties of hybrid species B. napus. The experiments involved determining the ratio of parental genes in hybrids, sequencing and methylation analysis of the promoter region of rDNA. Using Southern hybridization, it was revealed significant variability in the number of parental rDNA units between each variety. Data from sequence analysis were in good agreement with the results of Southern blot. Genetic recombination between parental rDNA units was revealed in one variety by DNA sequencing of promotor region. To study methylation, bisulfite sequencing was performed. It was found out that rDNA units of B. rapa origin have a higher value of methylation than units originated from B. oleracea.
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