A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis.

摘要

The genomic grapevine (Vitis vinifera L.) DNA extraction is difficult because of secondary metabolitesudthat interfere with DNA isolation procedures and subsequent applications. We developed a simple,udrapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogeninfectedudgrape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromideud(CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf materialudwithout employment of liquid nitrogen for initial tissue grinding. The protocol includedudpolyvinylpyrrolidone (PVP) to bind phenolic compounds, β-mercaptoethanol to inhibit the oxidation ofudpolyphenols, and a high concentration of NaCl (2.5 M) to increase the solubility of polysaccharides,udthus reducing their co-precipitation with DNA. Final DNA solution did not contain polysaccharides,udpolyphenols and other major contaminants. The purity of genomic DNA was confirmed by A260/280 andudA260/230 ratios calculated from the spectrophotometric readings. In addition, the quality of the DNAudextracted from asymptomatic, Oidium tuckeri- and Plasmopara viticola-infected leaves of V. vinifera L.udwas evaluated in polymerase chain reaction (PCR) analyses by using different set of primers to be ableudto amplify vegetal, fungal and bacterial DNA.