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Impact of G2 Enhancer on DNA extraction from recalcitrant soil core samples

ABSTRACT: Qualitative and quantitative evaluation of the impact of G2-Enhancer on DNA extraction from deep-soil samples

PROVIDER: PRJEB24676 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR2273870_1.fastq.gz	Fastqsanger.gz
	ERR2273870_2.fastq.gz	Fastqsanger.gz
	ERR2273880_1.fastq.gz	Fastqsanger.gz
	ERR2273880_2.fastq.gz	Fastqsanger.gz
	ERR2273871_1.fastq.gz	Fastqsanger.gz

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Quantitative and qualitative evaluation of the impact of the G2 enhancer, bead sizes and lysing tubes on the bacterial community composition during DNA extraction from recalcitrant soil core samples based on community sequencing and qPCR.

Project description:Soil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition. To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers from 1.00 to 2.20 m. Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition. Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier. These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples.

| S-EPMC6459482 | biostudies-literature

Phaeobacter sp. G2 strain:G2 | isolate:G2

Project description:Phaeobacter sp. G2 strain:G2 | isolate:G2 Genome sequencing

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Lysinibacillus sp. G2 strain:G2

Project description:Lysinibacillus sp. G2 strain:G2 Genome sequencing and assembly

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Kineococcus sp. G2 strain:G2

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Protein extraction in recalcitrant plant species

Project description:A possible approach for gel-based proteomic studies in recalcitrant woody plants, using phenol as the most suitable protein extraction method when working with tissues with high interfering compounds

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Impact of sand extraction on sediment prokaryote communities

Project description:Impact of sand extraction on sediment prokaryote communities

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Hypoxia-induced alterations of G2 checkpoint regulators

Project description:Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, in addition to DNA repair, genome stability is also controlled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can alter the DNA damage cell cycle checkpoints. Here, we show that hypoxia (24h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, flow cytometric bar-coding analysis of individual cells showed that the levels of several G2 checkpoint regulators were reduced in G2 phase cells after hypoxic exposure, in particular cyclin B1. These effects were accompanied by decreased Cyclin dependent kinase (CDK) activity in G2 phase cells after hypoxia. Furthermore, cells pre-exposed to hypoxia showed a longer G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (~0.03 % O2 20h and 0.2% O2 72h). These results demonstrate that the DNA damage G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors. We measured gene expression changes in U2OS cells after treatment with 0.2% hypoxia for 24 hours. The data were used in the exploration of hyopxia induced alterations in the G2 checkpoint.

2016-01-04 | E-GEOD-70530 | biostudies-arrayexpress

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