Efficiency of DNA extraction from the leaves of the common oak (Q. robur L.) and the needles of the Scots pine (P. sylvestris L.)
Rubrics: NATURAL SCIENCES AND FOREST
Authors:
1.
Voronezh State University of Forestry and Technologies named after G.F. Morozov
Russian Federation
Russian Federation
2.
Voronezh State University of Forestry and Technologies named after G.F. Morozov
(Department of forest inventory, forest taxation and forest management, Head of Department)
Voronezh, Voronezh, Russian Federation
Voronezh, Voronezh, Russian Federation
Type:
Article
Pages:
from 82
to 100
Status:
Published
Received:
01.06.2025
Accepted:
15.09.2025
Published:
25.09.2025
Subject area:
UDC 63
Language:
Russian
Keywords:
DNA extraction, extraction methods, Quercus robur (English oak), Pinus sylvestris (Scots pine), CTAB method, additional purification using Biolabmix columns, SKYSuper Plant Genomic DNA Kit, D-Plants, polysaccharides, polyphenols, optical density
Funding:
The study was carried out with the support of a grant from the Russian Science Foundation No. 24-16-20047, https://rscf.ru/project/24-16-20047/.
Abstract (English):
The extraction of high-quality DNA from tissues of woody plants, such as English oak (Quercus robur L.) and Scots pine (Pinus sylvestris L.), is challenging due to the high content of secondary metabolites (polyphenols, polysac-charides). These compounds co-extract with nucleic acids and inhibit downstream molecular genetic analyses. The aim of this work was a comparative evaluation of the efficiency of different DNA extraction methods (a modified CTAB protocol, a CTAB protocol with additional cleanup, and two commercial kits: the SKYSuper Plant Genomic DNA Kit and D-Plants) for obtaining DNA samples suitable for further research. The quality of the DNA preparations was as-sessed spectrophotometrically by the absorbance ratios A260/A280 (to assess protein contamination) and A260/A230 (to assess contamination by polysaccharides and polyphenols), electrophoretically (to visualize DNA integrity and the presence of impurities), and quantitatively. Functional suitability was tested via PCR amplification. For Scots pine nee-dles, the D-Plants kit demonstrated consistently high quality of the preparations (A260/A280 = 1.87 ± 0.33; A260/A230 = 1.95 ± 0.09) and 100% PCR efficiency. The SKYSuper kit showed the highest DNA yield but also signs of degradation (A260/A280 > 2.0) and low PCR efficiency (<50%). The classic CTAB method was the most economi-cal but required mandatory additional cleanup for polysaccharides. For English oak leaves, only the D-Plants kit yield-ed PCR-suitable DNA (80% efficiency), although the yield was very low (0.15 μg), resulting in a high cost per 1 μg of DNA. The other methods, including CTAB with cleanup, failed to produce a functional preparation (0% PCR efficien-cy). The D-Plants kit is the optimal solution for tasks requiring high-quality DNA (PCR, sequencing) for pine. For large-scale screening of pine, the classic CTAB method with subsequent cleanup is preferable. For English oak, none of the tested methods yielded a satisfactory result, indicating the need to develop specialized protocols including PVP pre-treatment steps.
The extraction of high-quality DNA from tissues of woody plants, such as English oak (Quercus robur L.) and Scots pine (Pinus sylvestris L.), is challenging due to the high content of secondary metabolites (polyphenols, polysac-charides). These compounds co-extract with nucleic acids and inhibit downstream molecular genetic analyses. The aim of this work was a comparative evaluation of the efficiency of different DNA extraction methods (a modified CTAB protocol, a CTAB protocol with additional cleanup, and two commercial kits: the SKYSuper Plant Genomic DNA Kit and D-Plants) for obtaining DNA samples suitable for further research. The quality of the DNA preparations was as-sessed spectrophotometrically by the absorbance ratios A260/A280 (to assess protein contamination) and A260/A230 (to assess contamination by polysaccharides and polyphenols), electrophoretically (to visualize DNA integrity and the presence of impurities), and quantitatively. Functional suitability was tested via PCR amplification. For Scots pine nee-dles, the D-Plants kit demonstrated consistently high quality of the preparations (A260/A280 = 1.87 ± 0.33; A260/A230 = 1.95 ± 0.09) and 100% PCR efficiency. The SKYSuper kit showed the highest DNA yield but also signs of degradation (A260/A280 > 2.0) and low PCR efficiency (<50%). The classic CTAB method was the most economi-cal but required mandatory additional cleanup for polysaccharides. For English oak leaves, only the D-Plants kit yield-ed PCR-suitable DNA (80% efficiency), although the yield was very low (0.15 μg), resulting in a high cost per 1 μg of DNA. The other methods, including CTAB with cleanup, failed to produce a functional preparation (0% PCR efficien-cy). The D-Plants kit is the optimal solution for tasks requiring high-quality DNA (PCR, sequencing) for pine. For large-scale screening of pine, the classic CTAB method with subsequent cleanup is preferable. For English oak, none of the tested methods yielded a satisfactory result, indicating the need to develop specialized protocols including PVP pre-treatment steps.
Keywords:
DNA extraction, extraction methods, Quercus robur (English oak), Pinus sylvestris (Scots pine), CTAB method, additional purification using Biolabmix columns, SKYSuper Plant Genomic DNA Kit, D-Plants, polysaccharides, polyphenols, optical density
DNA extraction, extraction methods, Quercus robur (English oak), Pinus sylvestris (Scots pine), CTAB method, additional purification using Biolabmix columns, SKYSuper Plant Genomic DNA Kit, D-Plants, polysaccharides, polyphenols, optical density
