28th Congress of SCTM

INFLUENCE OF DNA EXTRACTION METHODS ON DNA QUALITY, POLYPHENOL CONTENT AND PCR-HRM PERFORMANCE IN STACHYS SPECIES

Not scheduled

10m

Metropol Lake Resort

E-poster Organic chemistry, biochemistry and pharmaceutical chemistry

Speaker

Blagica Janeva (Institute of Chemistry, Faculty of Natural Sciences & Mathematics, Ss. Cyril and Methodius University in Skopje N. Macedonia)

Description

Species of the genus Stachys (Lamiaceae) are widely distributed medicinal and aromatic plants characterized by considerable morphological diversity and rich phytochemical composition. Molecular identification of Stachys species requires high-quality DNA. However, the presence of secondary metabolites, particularly polyphenols, may interfere with DNA isolation and downstream molecular analyses. This study compared three DNA extraction approaches: a cetyltrimethylammonium bromide (CTAB)-based protocol, the Edwards method based on sodium dodecyl sulfate (SDS), and a commercial QIAGEN DNA extraction kit.
Genomic DNA was isolated from dry Stachys samples and evaluated in terms of DNA yield, purity, polyphenol content, and suitability for quantitative PCR (qPCR) and PCR-high resolution melting (PCR-HRM) analysis. Species-specific primers targeting the ITS1 region were used for amplification and HRM analysis.
The Edwards protocol yielded the highest DNA concentration (248.3 ± 27 ng/µL), followed by the CTAB method (108.5 ± 16 ng/µL), while the commercial QIAGEN kit produced substantially lower DNA concentrations (52.5 ± 7 ng/µL). DNA purity (A260/A280) was relatively low for both the Edwards and CTAB methods (1.1) and slightly higher for the commercial kit (1.3). The concentration of co-extracted polyphenols was highest in Edwards extracts (22.6 ± 2.3 mg GAE/L), followed by CTAB extracts (7.1 ± 0.6 mg GAE/L), and lowest in DNA isolated using the commercial QIAGEN kit (4.8 ± 0.2 mg GAE/L).
Despite providing the highest DNA yield, the Edwards extracts exhibited pronounced qPCR inhibition, whereas DNA isolated using the CTAB protocol and the commercial kit showed efficient amplification. Additional purification of Edwards extracts with chloroform/isoamyl alcohol reduced polyphenol content (9.4 ± 0.7 mg GAE/L) and markedly improved qPCR performance, suggesting that co-extracted inhibitors, potentially including residual SDS, affected polymerase activity. PCR-HRM analysis produced comparable melting profiles among successfully amplified samples.
These findings demonstrate that DNA yield alone is not a reliable indicator of DNA suitability for molecular applications. Although the Edwards method enables rapid and efficient DNA recovery, additional purification may be required prior to PCR-based analyses of Stachys species.