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.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:376836 |
| Date | January 2018 |
| Creators | Pešková, Aneta |
| 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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