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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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