Polyphenolics free DNA isolation and optimization of PCR-RAPD for fennel (Foeniculum vulgare Mill.) from mature and young leaves

Abstract

Molecular analysis of fennel (Foeniculum vulgare Mill.) strictly relies on high yield, and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide (CTAB) protocol introducing several modifications such as inclusion of variable percentage of polyvinylpyrrolidone (PVP, 2 - 6%), different molarity of sodium chloride (NaCl, 1.5 - 4 M), activated charcoal (1 to 2%), and pre-heated buffer (65°C) with and without liquid N2 extraction for the mature leaves. In contrast, the CTAB protocol without any additional anti-oxidants using liquid N₂ extraction was performed for the fresh young leaf tissue. Optimization of polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD) conditions included 10X PCR buffer compositions such as (NH₄)₂SO₄ with 0.1% (v/v) Tween 20 over KCl buffer with and without 0.8% (v/v) Nonidet P40 and an ‘optimized’ buffer which contains KCl and (NH₄)₂SO₄, MgCl₂ (2.5 mM), Taq enzyme (1 to 1.5 U), annealing temperature of 37 and 42°C and PCR reaction volume of 10 and 25 μl. The results show that DNA isolated from fresh young leaves were superior in quality and quantity over mature (stored) leaves and was amenable to optimized PCR-RAPD conditions.

Keywords: Activated charcoal, cetyl trimethyl ammonium bromide (CTAB)-DNA, Foeniculum vulgare, NaCl, polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD), PCR buffer, polyphenolics, polyvinylpyrrolidone (PVP)