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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.

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.

In thesis was verified micromethod for isolation of plant DNA from different vegetable (onion and broccoli) and plant food products in quality for application in polymerase chain reaction (PCR). The micromethod allows isolation DNA using magnetic particles from crude lysates of cells obtained by direct homogenization of plant tissues. Various methods of processing homogenates were compared. Homogenization was performed by lysis buffer containing cetyltrimethylammonium bromide (CTAB). The effect of the organic extraction agents was tested (chloroform-octanol and isopropanol). DNA was purified from homogenates by reversible adsorption on magnetic particles (four different types of magnetic particles were tested). The quality of isolated DNA was verified by UV spectrophotometry. The amplificabilty of DNA was tested by polymerase chain reaction (PCR). Specific primers for plant ribosomal DNA (rDNA) were used. PCR products of lenght 700 and 220 bp were detected by agarose gel electrophoresis. Differences in yield and quality of DNA were depended on the homogenate processing and magnetic particles used. The proposed procedure with two magnetic particles was tested for the isolation DNA from plan food products (spreads). DNA was amplified in PCR. Micromethod is suitable for DNA analysis of foods.

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.

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.

Syftet med litteraturstudien är att sammanfatta vilken gensekvens som används vid genetisk barcoding av växter och hur väl metoden i fråga tillämpas i tre biologiska yrkesområden: dietanalyser i ekologin, analys av pollensporer i forensisk biologi samt analys av uråldrigt DNA (ancient DNA) i paleontologin. Vidare var det även av intresse att se hur genetisk barcoding kan användas i den gymnasiala undervisningen och hur väl den passar in med de svenska styrdokumenten för skolan. Hur elever har gynnats av den valda metoden samt vilka begränsningar som har uppstått har också berörts. Litteraturstudien baseras på vetenskapliga artiklar som har sökts fram med de nedan listade nyckelorden. Resultaten visar att en kombination av gensekvenser, däribland rbcL, matK, trnH-psbA och ITS, fungerar bäst vid identifiering av växter. I dagsläget är genetisk barcoding fortfarande i utvecklingsfasen, där metoden begränsas av antalet referenssekvenser i databaserna, vilket gör det svårt att utesluta morfologiska identifieringsmetoder i de tre yrkesområdena. Vid användning av barcoding i den gymnasiala undervisningen visar det sig att det stämmer väl överens med de svenska styrdokumenten och ökar elevers intresse för de naturvetenskapliga ämnena, då de kan bidra med värdefull forskning genom tillägg av referenssekvenser i databaserna. De största begränsningarna är att det blir ett stort arbetslass för läraren, att läraren i fråga måste vara bekväm med de olika laboratiska momenten och att skolan ska ha tillgång till nödvändig apparatur. / The purpose of the literature study is to conclude which gene sequences are being used in DNA barcoding on plants and how the method in question is being used in three different biological occupations: diet analysis in ecology, analysis of pollen in forensics and analysis of ancient DNA (aDNA) in paleontology. Further it was also of interest to study how DNA barcoding can be used in high school settings and how the method correlates with the Swedish curriculum. How pupils have benefited from the chosen method and what limitations have arisen have also been touched upon. This literature study is based on scientific articles that have been sought with the keywords listed below. The results show that a combination of gene sequences, including rbcL, matK, trnH-psbA and ITS, works best in plant identification. At present, genetic barcoding is still in the developmental phase, where the method is limited by the number of reference sequences in the databases, which makes it difficult to exclude morphological-based methods in the three occupational fields. When using barcoding in upper secondary education it turns out that it’s in good agreement with the Swedish curriculum and increases the students' interest in the scientific subjects, since they can contribute with genuine research when adding reference sequences in the databases. The main limitations are the workload for the teacher, the teacher in question must be comfortable with the different laboratory steps and that the school must have access to necessary equipment.