ORIGINAL ARTICLE A novel method for the isolation of DNA from intracellular bacteria, suitable for genomic studies Sara Epis & Alberto M. Luciano & Federica Franciosi & Chiara Bazzocchi & Elena Crotti & Dario Pistone & Claudio Bandi & Davide Sassera Received: 7 April 2010 / Accepted: 27 April 2010 / Published online: 19 May 2010 # Springer-Verlag and the University of Milan 2010 Abstract We present a protocol for the preparation of DNA suitable for genomic studies, starting from uncultured intracellular bacteria. The study was conducted on Midi- chloria 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. Keywords MDA . qPCR . Ixodes ricinus . Midichloria mitochondrii Introduction Midichloria mitochondrii is an endosymbiotic bacterium of the order Rickettsiales, found in the hard tick Ixodes ricinus (Beninati et al. 2004; Sassera et al. 2006). This bacterium is intracellular, and has not yet been cultured in cell-free medium, as is the case for all Rickettsiales. A unique feature of this bacterium is its localization: electron microscopy shows that these bacteria reside, and probably multiply, inside the mitochondria of tick ovarian cells. The bacterium is located in the intermembrane space of the mitochondria, where its multiplication apparently leads to the consumption of the mitochondrial matrix (Sacchi et al. 2004). The prevalence of M. mitochondrii in wild-collected females of I. ricinus is 100% (Lo et al. 2006). Quantitative real-time PCR (qPCR) shows that the ovary of I. ricinus contains a very high load of M. mitochondrii: a single tick ovary can contain several millions of genome copies of this symbiont, and up to 10 5 –10 6 genome copies can be detected in single eggs (Sassera et al. 2008). Information on the biology of M. mitochondrii is scarce, and only a limited number of gene sequences from this S. Epis is on leave from Università di Camerino, Dipartimento di Medicina Sperimentale e Sanità Pubblica S. Epis : C. Bazzocchi : D. Pistone : C. Bandi : D. Sassera (*) Sezione di Patologia Generale e Parassitologia, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy e-mail: davide.sassera@unimi.it A. M. Luciano : F. Franciosi Sezione di Anatomia ed Istologia Veterinaria, Dipartimento di Scienze Animali, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy E. Crotti Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Facoltà di Agraria, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy Ann Microbiol (2010) 60:455–460 DOI 10.1007/s13213-010-0063-y