TY - JOUR

T1 - Easy and Effective Method for Extracting and Purifying Wolbachia Genomic DNA

AU - Andreenkova, Olga V.

AU - Shishkina, Olga D.

AU - Klimenko, Alexandra I.

AU - Korenskaia, Aleksandra E.

AU - Bobrovskikh, Margarita A.

AU - Shatskaya, Natalja V.

AU - Vasiliev, Gennady V.

AU - Gruntenko, Nataly E.

N1 - Funding Information: This research was funded by the Russian Science Foundation, #21-14-00090. The maintenance of experimental D. melanogaster strains was carried out in the Drosophila collection fund of the Institute of Cytology and Genetics SB RAS and was supported by BP #FWNR-2022-0019 of the Ministry of Science and Higher Education of the Russian Federation. Publisher Copyright: © 2022 by the authors.

PY - 2022/12

Y1 - 2022/12

N2 - A number of methods for extracting the DNA of maternally inherited obligate intracellular bacteria Wolbachia from an insect host and its subsequent purification have been described in previous scholarship. As Wolbachia 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 Wolbachia DNA from Drosophila melanogaster with a purity sufficient for genomic sequencing. Our method involves the centrifugation of homogenized flies or just their ovaries, as the most Wolbachia-enriched tissue, followed by the filtration of homogenate and extraction of DNA using a modified version of the Livak buffer protocol. The proportion of Wolbachia 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 D. melanogaster lines infected with two different Wolbachia strains, the proportion was at least 68 and 94%, respectively.

AB - A number of methods for extracting the DNA of maternally inherited obligate intracellular bacteria Wolbachia from an insect host and its subsequent purification have been described in previous scholarship. As Wolbachia 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 Wolbachia DNA from Drosophila melanogaster with a purity sufficient for genomic sequencing. Our method involves the centrifugation of homogenized flies or just their ovaries, as the most Wolbachia-enriched tissue, followed by the filtration of homogenate and extraction of DNA using a modified version of the Livak buffer protocol. The proportion of Wolbachia 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 D. melanogaster lines infected with two different Wolbachia strains, the proportion was at least 68 and 94%, respectively.

KW - Drosophila melanogaster

KW - genome DNA extraction

KW - Wolbachia

KW - Drosophila melanogaster/genetics

KW - Symbiosis

KW - Animals

KW - DNA

KW - Wolbachia/genetics

KW - Chromosome Mapping

KW - Sequence Analysis, DNA

UR - http://www.scopus.com/inward/record.url?scp=85143616861&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/4e20c6cc-9bce-36b5-a218-4000fa176edc/

U2 - 10.3390/ijms232315315

DO - 10.3390/ijms232315315

M3 - Article

C2 - 36499640

AN - SCOPUS:85143616861

VL - 23

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 23

M1 - 15315

ER -