The purpose of this study was to identify a reliable DNA extraction protocol to use on 25-year-old powdery mildew specimens from the reference collection VPRI in order to produce high quality sequences suitable to address taxonomic phylogenetic questions. We tested 13 extraction protocols and two library preparation kits and found the combination of the E.Z.N.A.® Forensic DNA kit for DNA extraction and the NuGen Ovation® Ultralow System library preparation kit was the most suitable for this purpose.
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ACS Synth Biol
December 2024
Proteo-Science Center, Ehime University, 2-5 Bunkyo, Matsuyama, Ehime 790-8577, Japan.
Cell-free systems, which can express an easily detectable output (protein) with a DNA or mRNA template, are promising as foundations of biosensors devoid of cellular constraints. Moreover, by encasing them in membranes such as natural cells to create artificial cells, these systems can avoid the adverse effects of environmental inhibitory molecules. However, the bacterial systems generally used for this purpose do not function well at ambient temperatures.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
Here, we present a protocol for the isolation and detection of Phytophthora oospores directly from soil samples. Our method incorporates a novel technique for isolating Phytophthora oospores using filter pouches and an improved DNA extraction procedure specifically designed for oospores. While we have primarily developed this protocol for detecting P.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
The James Hutton Institute, Dundee, UK.
We describe a protocol to amplify DNA barcodes of known and unknown taxa of Phytophthora and related plant pathogenic oomycetes from a range of environments. The methods focus on sampling pathogen propagules from water using in situ sampling and filtration equipment and buffers that enable efficient storage and DNA extraction for later downstream processing.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USA.
Transcriptional regulation allows cells to execute developmental programs, maintain homeostasis, and respond to intra- and extracellular signals. Central to these processes are promoters, which in eukaryotes are sequences upstream of genes that bind transcription factors (TFs) and which recruit RNA polymerase to initiate mRNA synthesis. Valuable tools for studying promoters include reporter genes, which can be used to indicate when and where genes are activated.
View Article and Find Full Text PDFVet Sci
December 2024
Camel Research Center, King Faisal University, 400 Al-Ahsa, Hofuf 31982, Saudi Arabia.
Currently, bacterial classification at the species level relies on the 95-96% average nucleotide identity (ANI) value that is known to be equivalent to a 70% digital DNA-DNA hybridization (dDDH) value. However, during the routine identification of bacteria in the uteri of camels with a history of conception failure, we found that four out of the seven strains (2298A, 2569A, 2652, 2571B, 1103A, 2571A, and 335C) could not be assigned to any valid species. Furthermore, a 70% dDDH value did not correspond to a 95-96% ANI value in strain 2569A.
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