An Optimized and Rapid DNA Extraction Method From Leaves of Grapevine Suitable for PCR-DGGE Based Analysis

Abstract

Molecular techniques and genetic studies require a fair amount of DNA of high quality in order to produce reliable and clear results (Li et al., 2007). In grapevine, the yield and quality of DNA can be significantly affected by secondary metabolites such as polyphenols, polysaccharides and tannins, that may be consistently abundant during the different stages of leaf development (Iandolino et al., 2004). The development of a simple, rapid and reliable method for the extraction of genomic DNA from grape leaves collected at several stages during development, as well as from healthy grape leaves and leaves infected by pathogenic microorganisms, was the main objective of this study. The protocol reported, based on a modified cetyltrimethylammonium bromide (CTAB) extraction procedure (Doyle and Doyle, 1990), allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone to bind phenolic compounds, β-mercaptoethanol to inhibit the oxidation of polyphenols, and a high concentration of NaCl (2.5 M) to increase the solubility of polysaccharides, thus, reducing their co-precipitation with DNA. Final DNA solution did not contain polysaccharides, polyphenols and other major contaminants. The quality of the DNA extracted with the above protocol was first evaluated for its accessibility for polymerase chain reaction (PCR); genomic DNA from healthy and Plasmopara viticola infected leaves of Vitis vinifera was successfully amplified by traditional PCR. Subsequently, the same DNA solution was used for amplification with a primer pair specific for PCR-denaturing gradient gel electrophoresis (DGGE), containing a GC-clamp at the 5′-end of the forward oligonucleotide. PCR produced amplicons with the expected molecular size (200 bp) suitable to be analyzed by means of a denaturing containing polyacrilamide gel for the bacterial ecology composition.