METODY IZOLACE DNA Z ČERSTVÉ DŘEVNÍ HMOTY PRO ÚČELY MOLEKULÁRNÍ DIAGNOSTIKY V OVOCNÁŘSTVÍ

DNA was isolated from fresh wood tissue of apricot tree trunks (Prunus armeniaca L.) using three different commercial kits (manufacturers GeneAll, Zymo Research, Wizbiosolutions) and two methods of mechanical homogenization of wood chips (liquid nitrogen and mortar and pestle vs. ball mill). The aim of this study was to improve the yield and quality of DNA extracted from woody tissue of fruit tree for subsequent molecular biological analyses. We focused on two key factors affecting the quality of DNA isolated from wood: (i) the effect of drill bit diameter on the size and quantity of wood chips and their suitability for subsequent processing, and (ii) a comparison of mechanical homogenization techniques and DNA extraction protocols in terms of DNA fragmentation. The lowest Ct values (real-time PCR targeting a chloroplast gene) and highest reproducibility were achieved using a 1.5 mm diameter drill bit, while a 4 mm diameter drill bit proved to be the most effective for rapid material collection. Homogenization in liquid nitrogen resulted in higher DNA yields for the GeneAll and Wizbiosolutions kits compared to ball mill homogenization, whereas the Zymo Research kit performed better with ball mill homogenization. Amplification of chloroplast genes rbcL (599 bp) and matK (1292 bp) as well as the fungal-specific rDNA region (specifically the ITS region) (210–230 bp) confirmed differences in DNA quality between individual protocol combinations. The longest fragment (matK) was successfully amplified in all samples only when DNA was extracted using the Wizbiosolutions kit. Overall, the best results in terms of DNA yield, purity, and amplifiability were obtained with the Wizbiosolutions kit.