African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12368

Full Length Research Paper

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

Sandra Lo Piccolo1, Antonio Alfonzo1, Gaetano Conigliaro1, Giancarlo Moschetti1, Santella Burruano1 and Amalia Barone2*
1Dipartimento Demetra, Università di Palermo, Viale delle Scienze 4, 90128 Palermo, Italy. 2Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università Federico II di Napoli, Via Università 100, 80055 Portici, Italy.  
Email: [email protected]

  •  Accepted: 16 December 2011
  •  Published: 05 June 2012

Abstract

The genomic grapevine (Vitis vinifera L.) DNA extraction is difficult because of secondary metabolites that interfere with DNA isolation procedures and subsequent applications. We developed a simple, rapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogen-infected grape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromide (CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone (PVP) 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 purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings. In addition, the quality of the DNA extracted from asymptomatic, Oidium tuckeri- and Plasmopara viticola-infected leaves of V. vinifera L. was evaluated in polymerase chain reaction (PCR) analyses by using different set of primers to be able to amplify vegetal, fungal and bacterial DNA.

 

Key words: Vitis vinifera L., DNA extraction, PCR, fungi, bacteria.

Abbreviation

CTAB, Cetyl trimethylammonium bromide; EDTA, ethylenediaminetetraacetic acid; PCR, polymerase chain reaction; PVP, polyvinylpyrrolidone.