Determination of the Effects of Different DNA Isolation Methods on Quantity and Quality
Öz
Objective: This study compared the effects of different DNA extraction methods (CTAB, phenol-chloroform, silica columns, magnetic beads) on the DNA yield and purity obtained from maize (Zea mays L.), bean (P. vulgaris L.), oak (Q. robur L.) and scots pine (P. sylvestris L.).
Materials and Methods: In this study, young apical leaves (the most distal fully expanded leaves) of maize, bean, oak and scots pine were used. For each species, 10 leaf tissue samples were collected from young apical leaves during the vegetation season of the year 2024 from plantations located at different sites within the Bayramiç district of Çanakkale province. Leaf tissue samples were collected during the vegetation season of the year 2024, specifically during the early phenological stages of each species. For maize and bean, samples were obtained in the early vegetative growth phase, when the apical leaves were fully expanded but still young and metabolically active. For oak and Scots pine, sampling was conducted during the early spring, shortly after bud burst, when newly emerged apical leaves were in the initial phase of leaf expansion. Young apical leaves were preferred as they contain fewer secondary metabolites and phenolic compounds, which can interfere with DNA extraction and downstream molecular applications. This sampling strategy ensured the collection of high-quality tissue suitable for genomic DNA isolation. DNA isolation was performed using four different methods: CTAB, phenol-chloroform, silica-based columns, and magnetic beads. These methods were compared in terms of their suitability for genetic analyses specific to each plant species.
Results: Using the phenol-chloroform method, a notably high DNA yield was obtained from P. sylvestris L. samples (371.75 ng/µL). Similarly, Q. robur L. also exhibited high yield (352.00 ng/µL), suggesting that these species are particularly compatible with this method. The CTAB method also yielded successful results. P. sylvestris L. achieved a high DNA yield (323.75 ng/µL), indicating that this species' genetic material is compatible with the CTAB method. A DNA yield of 308.00 ng/µL was observed for Q. robur L., which may necessitate additional steps for protein removal. The silica column method yielded DNA samples of a lower quantity than the other three methods. For P. sylvestris L. and Q. robur L., yields of (230.75 ng/µL) and (222.25 ng/µL), respectively, were recorded. Lower yields were observed in P. vulgaris L. (171.75 ng/µL) compared to Zea mays L. (211.75 ng/µL). The diminished DNA recovery may be attributable to the constrained binding capacity of silica columns or inadequate initial material. In the magnetic beads method, DNA yields for P. sylvestris L. (206.25 ng/µL), Q. robur L. (201.75 ng/µL), Zea mays L. (173.50 ng/µL) and P. vulgaris L. (151.75 ng/µL) were the lowest among all methods.
Conclusion: This study compares DNA isolation methods including CTAB, phenol-chloroform, silica columns, and magnetic beads for Zea mays L., P. vulgaris L., Q. robur L., and P. sylvestris L. The findings reveal that the differences in DNA yields and purity depend on the plant species and tissue characteristics. The phenol-chloroform method provided the highest DNA yields and purity across all plant species. This method excels in obtaining DNA free from proteins, polysaccharides, and phenolic compounds, ensuring high molecular integrity. The results demonstrate that Q. robur L. and P. sylvestris L., which are rich in lignin and phenolic compounds, had the highest yields and preserved molecular integrity. This supports the use of the phenol-chloroform method in molecular analyses requiring high-quality DNA.
Anahtar Kelimeler
DNA isolation, CTAB, Phenol-chloroform, Silica columns, Magnetic beads
Kaynakça
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