A number of methods for extracting the DNA of maternally inherited obligate intracellular bacteria from an insect host and its subsequent purification have been described in previous scholarship. As is present in the hosts' organisms in rather low quantities, these techniques used to be quite labor-intensive. For this paper, we analyzed them in detail, searched for a possibility to simplify and accelerate the protocol, and proposed an easy and effective method for isolating DNA from with a purity sufficient for genomic sequencing. Our method involves the centrifugation of homogenized flies or just their ovaries, as the most -enriched tissue, followed by the filtration of homogenate and extraction of DNA using a modified version of the Livak buffer protocol. The proportion of DNA in the total DNA was quantified based on the results of sequencing with the use of the Illumina MiSeq platform and a pipeline of bioinformatic analysis. For the two analyzed lines infected with two different strains, the proportion was at least 68 and 94%, respectively.
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| http://dx.doi.org/10.3390/ijms232315315 | DOI Listing |
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