A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores

Abstract

 We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene.