A novel method for the isolation of DNA from intracellular bacteria, suitable for genomic studies

Abstract

We present a protocol for the preparation of DNA suitable for genomic studies, starting from uncultured intracellular bacteria. The study was conducted on Midichloria mitochondrii, the intramitochondrial symbiont of the tick Ixodes ricinus. Single oocytes, which carry a high load of symbionts, were microdissected from the ovary of a semi-engorged tick specimen. After short incubation of oocytes in hypotonic medium, the cytoplasm that extruded from the cells was collected and used as template for multiple displacement amplification (MDA). Eight MDA preparations were examined with real-time PCR, targeted on seven loci of M. mitochondrii (to assess the uniformity of the genome amplification) and on four loci of I. ricinus (to select DNA preparations showing minimal presence of host DNA). The two MDA products that presented minimal biases in terms of symbiont genes and minimal host-DNA presence were pooled and cloned in a plasmid library. Fifty clones were then sequenced. Only 6% of the sequences were of arthropod origin, while the vast majority could be inferred to have originated from M. mitochondrii. In summary, the developed protocol allowed us to generate micrograms of symbiont DNA, with minimal biases in genome representation and minimal presence of host DNA. © Springer-Verlag and the University of Milan 2010.