A New Approach to DNA Isolation from Dileptid Ciliates (Ciliophora: Litostomatea: Rhynchostomatia)

Abstract

Molecular diagnostic methods have been used to supplement morphological methods in taxonomy. In this study, we used molecular methods to diagnose populations of dileptid ciliates, which commonly occur in terrestrial and semi-terrestrial habitats. Molecular investigations based on the comparison of DNA sequences have been carried out with a limited number of ciliate species due to insufficient genomic DNA for PCR because of their small population size and difficulties to maintain monocultures. The present study shows that there is a loss of DNA with conventional methods and proposes a novel approach: cells (stored at -20 degrees C or fresh) are directly subjected to PCR, without being processed by any chemical treatment. The small subunit ribosomal DNA (SSU rDNA) (18S rRNA gene) of dileptid ciliates was successfully amplified by direct PCR, without DNA extraction, for single-cell and multi-cell samples. This method has been found to be simpler and especially useful for species that are rare and have small population sizes.