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
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 -