Optimization of Extraction Buffer Composition and Incubation Time for DNA Isolation from Vitis spp. Using a Matrix Experimental Design

Abstract

Grapevine tissues (Vitis spp.) are rich in various phenolic compounds and polysaccharides, which complicates the isolation of dsDNA for molecular analysis. In this study, 25 different DNA extraction buffers were developed and tested using a six-factor matrix method with five levels of variation. An optimized buffer based on 100 mM Tris-HCl (pH 8.0) was developed, containing 1% (m/v) CTAB, 1% (m/v) PVP, 5% (v/v) β-mercaptoethanol, 30 mM Na2EDTA, 1.0 M NaCl, and 60 min of incubation. The protocol allowed us to obtain highquality DNA (187–305 ng/μL, OD260/OD280 = 1.80–1.88) suitable for PCR from five grape varieties: ‘Chardonnay’, ‘Kober 5BB’, ‘Shine Muscat’, ‘Selection Oppenheim 4’, and ‘Fercal’, grown in vitro. This universal buffer improves the reproducibility of results in studies of genetic diversity, pathogen detection, and breeding.