Easy and Effective Method for Extracting and Purifying Wolbachia Genomic DNA

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 l...

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Bibliographic Details
Published in:International journal of molecular sciences 2022-12, Vol.23 (23), p.15315
Main Authors: Andreenkova, Olga V, Shishkina, Olga D, Klimenko, Alexandra I, Korenskaia, Aleksandra E, Bobrovskikh, Margarita A, Shatskaya, Natalja V, Vasiliev, Gennady V, Gruntenko, Nataly E
Format: Article
Language:English
Subjects:
Animals
Bacteria
Centrifugation
Chromosome Mapping
Communication
Deoxyribonucleic acid
DNA
Drosophila melanogaster
Drosophila melanogaster - genetics
Drosophila melanogaster - microbiology
Fruit flies
genome DNA extraction
Genomes
Genomics
Insects
Mitochondrial DNA
Mosquitoes
Ovaries
Pore size
Sequence Analysis, DNA
Symbiosis
Wolbachia
Wolbachia - genetics
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Summary: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.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms232315315