Bacterial DNA Extraction Using Individual Enzymes and Phenol/Chloroform Separation

Wright, Mitchell Henry; Adelskov, Joseph; Greene, Anthony Carlson

doi:10.1128/jmbe.v18i2.1348

Cited by 85 publications

(49 citation statements)

References 8 publications

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“…Unlike other African countries where further introduction events (i.e. T11, T12 and T13) within the 3 rd wave of the 7PET have been reported [15], it is noteworthy that isolates from eastern DRC all belonged only to the T10 introduction event. These results suggest that these T10 isolates have firmly established themselves in the Congolese Albertine rift, becoming an autonomous source of endemic, sporadic and epidemic cholera in the eastern DRC sub-region.…”

Section: Discussionmentioning

confidence: 92%

“…The V. cholerae O1 global phylogeny including data from Uganda [15], Tanzania [49], DRC, Central African Republic, and Zambia [5] confirms that the ST515 sub-clade has now spread to several regions of Central and Eastern Africa, including western provinces of DRC up to the Atlantic coast. Whereas the reason why only the ST515 expands so widely, and not the ST69 sub-clade, remains unknown, the hypothesis is that the higher genetic diversity among ST515 isolates results from a high mutation frequency, which favors their adaptation to changing environmental conditions.…”

Section: Discussionmentioning

confidence: 93%

“…V. cholerae isolates were cultured overnight in 10 ml Luria-Bertani broth. DNA was isolated using the phenol chloroform protocol [15]. DNA was quantified using the Nanodrop® and the Qubit® fluorometric quantitation (Thermo Fisher Scientific, Asse, Belgium) and normalized to 0.2 ng/µl.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Genomic analysis of pathogenic isolates ofVibrio choleraefrom eastern Democratic Republic of the Congo (2014-2017)

Irenge

Ambroise

Mitangala³

et al. 2019

Preprint

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AbstractBackgroundOver the past recent years, Vibrio cholerae has been associated with outbreaks in Sub Saharan Africa, notably in Democratic Republic of the Congo (DRC). This study aimed to determine the genetic relatedness of isolates responsible for cholera outbreaks in eastern DRC between 2014 and 2017, and their potential spread to bordering countries.Methods/Principal findingsPhenotypic analysis and whole genome sequencing (WGS) were carried out on 78 clinical isolates of V. cholerae associated with cholera in eastern provinces of DRC between 2014 and 2017. SNP-based phylogenomic data show that most isolates (73/78) were V. cholerae O1 biotype El Tor with CTX-3 type prophage. They fell within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and were contained in the introduction event (T)10 in East Africa. These isolates clustered in two sub-clades corresponding to Multiple Locus Sequence Types (MLST) profiles ST69 and the newly assigned ST515, the latter displaying a higher genetic diversity. Both sub-clades showed a distinct geographic clustering, with ST69 isolates mostly restricted to Lake Tanganyika basin and phylogenetically related to V. cholerae isolates associated with cholera outbreaks in western Tanzania, whereas ST515 isolates were disseminated along the Albertine Rift and closely related to isolates in South Sudan, Uganda, Tanzania and Zambia. Other V. cholerae isolates (5/78) were non-O1/non-O139 without any CTX prophage and no phylogenetic relationship with already characterized non-O1/non-O139 isolates.Conclusions/SignificanceCurrent data confirm the association of both DRC O1 7PET (T)10 sub-clades ST69 and ST515 with recurrent outbreaks in eastern DRC and at regional level over the past 10 years. Interestingly, while ST69 is predominantly a locally endemic sequence type, ST515 became adaptable enough to expand across DRC neighboring countries.Author’s summaryCholera is a severe diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. After originating in Asia, the disease spread across sub-Saharan Africa, notably Democratic Republic of the Congo. The aim of our study was to assess the transmission pattern of V. cholerae strains prevailing in eastern DRC, and determine their genetic relatedness to strains from other African countries and other parts of the world. Between 2014 and 2017, we isolated V. cholerae from fecal samples of patients with acute diarrhea in eastern DRC, and subsequently examined the DNA of the bacteria. The results show that they all clustered in two genetic groups (ST69 and ST515) falling within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and T10 introduction event in East Africa. The genetic signature of ST515 may be involved in its adaptation to environmental conditions found in eastern DRC, and contribute to its extended geographic distribution. Indeed, unlike the locally endemic ST69, ST515 is spreading extensively through DRC cross-border countries such as South Sudan, Tanzania, Uganda and Zambia. This plainly justifies a regional strategy to strengthen the fight against cholera in eastern Africa.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 93%

See 1 more Smart Citation

Genomic analysis of pathogenic isolates ofVibrio choleraefrom eastern Democratic Republic of the Congo (2014-2017)

Irenge

Ambroise

Mitangala³

et al. 2019

Preprint

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“…Genomic DNA of M. morganii strains MM 1, MM 4, and MM 190 was extracted by phenol chloroform method (Wright et al, 2017). The quantity and quality control of extracted DNA was evaluated using a NanoPhotometer P 300 (Implen, Germany) and by electrophoresis on a 1% agarose gel, respectively.…”

Section: Methodsmentioning

confidence: 99%

Comparative Genome Analysis of Uropathogenic Morganella morganii Strains

Миннуллина

Pudova

Shagimardanova

et al. 2019

Front. Cell. Infect. Microbiol.

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Morganella morganii is an opportunistic bacterial pathogen shown to cause a wide range of clinical and community-acquired infections. This study was aimed at sequencing and comparing the genomes of three M. morganii strains isolated from the urine samples of patients with community-acquired urinary tract infections. Draft genome sequencing was conducted using the Illumina HiSeq platform. The genomes of MM 1, MM 4, and MM 190 strains have a size of 3.82–3.97 Mb and a GC content of 50.9–51%. Protein-coding sequences (CDS) represent 96.1% of the genomes, RNAs are encoded by 2.7% of genes and pseudogenes account for 1.2% of the genomes. The pan-genome containes 4,038 CDS, of which 3,279 represent core genes. Six to ten prophages and 21–33 genomic islands were identified in the genomes of MM 1, MM 4, and MM 190. More than 30 genes encode capsular biosynthesis proteins, an average of 60 genes encode motility and chemotaxis proteins, and about 70 genes are associated with fimbrial biogenesis and adhesion. We determined that all strains contained urease gene cluster ureABCEFGD and had a urease activity. Both MM 4 and MM 190 strains are capable of hemolysis and their activity correlates well with a cytotoxicity level on T-24 bladder carcinoma cells. These activities were associated with expression of RTX toxin gene hlyA , which was introduced into the genomes by a phage similar to Salmonella phage 118970_sal4.

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“…Akan tetapi untuk mendapatkan hasil yang optimal, banyak peneliti memilih fase pertumbuhan tertentu untuk proses ekstraksi. DNA dari mikroorganisme bisa diperoleh dari setiap titik fase pertumbuhan bakteri, akan tetapi populasi sel dan keberadaan metabolit primer maupun sekunder lainnya bisa mempengaruhi jumlah dan kualitasnya (Wright et al, 2017). Selain itu, setiap mikroorganisme, termasuk bakteri, memiliki kompleksitas molekuler sendiri dengan kondisi media tumbuh yang berbeda-beda sehingga memungkinkan kondisi optimumnya berbeda dengan yang lainnya (Utnes et al, 2015).…”

Section: Hasil Dan Pembahasanunclassified

Optimasi Ekstraksi DNA Genomik Probiotik Lactobacillus plantarum IIA-1A5 dari Daging Sapi Peranakan Ongole untuk Sekuensing Genom Utuh

Budiman¹,

Arief²,

Yusuf³

2018

JIPTHP

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Lactobacillus plantarum IIA-1A5 is an Indonesian probiotic bacterium isolated from Ongole grade cattle. This strain has demonstrated series of benefical properties and promising to be further applied in development of functional foods. Attempts to further exploit this strain require comprehensive understanding on its molecular properties, in particular is the genes responsible for its probiotic properties. Whole genome sequencing (WGS) is currently an affordable tool to fully decipher genetic organization of the bacteria, yet often limited by the requirement of high amount of high quality of genomic DNA of sample. This study aims to develop an optimum method for obtaining high quality genomic DNA of L. plantarum IIA-1A5 in sufficient amount of WGS study. To address, genomic DNA of L. plantarum IIA-1A5 was extracted from growth phase of adaptation, exponential and stationary. The result showed that exponential and stationary phases were able to yield genomic DNA higher than minimum requirement for WGS. Nevertheless, the quality of genomic DNA from both phases were considerably low, according to their 260/280 and 260/230 ratio. The worst quality was found on 260/280 ratio referring to the presence of high amount of proteinaceous compounds in the sample. Further optimization was done on genomic DNA from stationary phase by adding proteinase K at various incubation time. It showed that the incubation for 7.5 to 10 hours yielded acceptable purity and amount of genomic DNA for WGS. In addition, we also observed a trade-off phenomenon of yield-purity genomic DNA of L. plantarum IIA-1A5. Altogether, the optimized method here should contribute to further molecular studies of this probiotic strain.

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Bacterial DNA Extraction Using Individual Enzymes and Phenol/Chloroform Separation

Cited by 85 publications

References 8 publications

Genomic analysis of pathogenic isolates ofVibrio choleraefrom eastern Democratic Republic of the Congo (2014-2017)

Genomic analysis of pathogenic isolates ofVibrio choleraefrom eastern Democratic Republic of the Congo (2014-2017)

Comparative Genome Analysis of Uropathogenic Morganella morganii Strains

Optimasi Ekstraksi DNA Genomik Probiotik Lactobacillus plantarum IIA-1A5 dari Daging Sapi Peranakan Ongole untuk Sekuensing Genom Utuh

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