Direct amplification of DNA from fresh and preserved ectomycorrhizal root tips

Abstract

Methods are described by which DNA can be amplified directly from ectomycorrhizal root tip homogenates of a variety of plant species ( Picea mariana (black spruce), Betula papyrifera (paper birch), Populus tremuloides (trembling aspen) and Alnus sp.(alder)), including root tips that have been preserved in RNA Later (Ambion, Austin, TX). In most cases for extracts and homogenates diluted 10-fold prior to PCR, and in all cases for 100-fold dilutions, direct amplification of DNA from fresh root tip homogenates yielded as many or more ng of PCR amplicon (fungal ITS region) than amplification of DNA extracted from the same tips using a commercial kit or a manual ethanol precipitation-based method. For alder root tip extracts diluted 10-fold, the commercial kit method yielded more ng of PCR amplicon than 10-fold diluted, although direct use of homogenates still resulted in amplification in all tips tested. We also demonstrate consistent amplification of DNA from homogenates of birch, spruce and aspen ectomycorrhizal root tips preserved for 4 months in RNA Later.