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.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:295653 |
| Date | January 2017 |
| Creators | Gaňová, Martina |
| Contributors | Rittich, Bohuslav, Kovařík, Aleš |
| Publisher | Vysoké učení technické v Brně. Fakulta chemická |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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