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Nanostructured Cellular Biomolecules and Their Transformation in Context of Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
Hybridization techniques display a wide scope of application, including DNA–DNA hybridization, polymerase chain reaction (PCR), southern blots, northern blots, forensic DNA testing, and in medical science for diagnostic purpose. DNA–DNA hybridization is a method utilized to measure the extent of genetic similarity between two organisms. For this, labeled DNA sequence of known organisms is incubated with an unknown DNA sequence to allow formation of hybrid double-stranded DNA. Hybridized sequence with great similarity will bind strongly and dissociates only at higher temperatures compared to dissimilar sequence. This method is used as a taxonomic gold standard for species delineation in Archaea and bacteria. DNA–DNA hybridization similarity below 70% signifies distinct relation between organisms. However, this process being laborious and tedious with high chance of error, alternative methods are being researched (Meier-Kolthoff et al. 2013). Fluorescence in situ hybridization is a method that utilizes fluorescent probes to detect specific DNA sequence. This technique has wide application for identification of species, gene, RNA (mRNA), tumor, or cancerous cells. High-sensitivity detection, simultaneous assay of multiple species, automated data collection, and analysis have made this method a foremost biological assay (Levsky and Singer 2003).
Taxonomic, metabolic traits and species description of aromatic compound degrading Indian soil bacterium Pseudomonas bharatica CSV86T
Published in Journal of Environmental Science and Health, Part A, 2023
Balaram Mohapatra, Prashant S. Phale
16S rRNA gene (1550 bp, accession: MN866057) was PCR amplified from genomic DNA of strain CSV86T using custom-synthesized primers (16SF: 5′-TCAGCATCACAAGTGACTATACGAGCAA-3′, 16SR: 5′-ACCCAGTTAAGAGCTGTTACCGTATCGTC-3′) designed by OligoEvaluatorTM (Sigma-Aldrich). Individual sequences were edited and assembled by BioEdit version 7.1.11, subjected to similarity search in NCBI BLASTN and against validly described members of the EzBioCloud valid-taxa server (http://www.ezbiocloud.net/). Multiple alignments and phylogenetic tree construction were performed with best matches of Pseudomonas type strains using CLUSTAL W package and MEGA 7.0, respectively. All algorithms, neighbor-joining (NJ), maximum-likelihood (ML) and minimum-evolution (ME) methods were employed to test the robustness of the trees with 1000 bootstrap resampling. High quality genomic DNA of CSV86T was sequenced through PacBio RS II (P4-C2) technology. For genome assembly, a hybrid assembly method was employed, where the Roche 454 GS (FLX Titanium) data[7] was merged with the PacBio reads in Canuv1.8 assembler to obtain/yield a high-quality advanced genomic draft (accession: AMWJ02000000). Functional annotation of the genome of strain CSV86T was performed using Rapid Annotation using Subsystem Technology (RAST-tk: https://rast.nmpdr.org/) and Genoscope (http://www.genoscope.cns.fr/agc/microscope/home/). Various genomic methods of taxonomic analyses such as multi-locus sequence analysis (MLSA) involving four conserved house-keeping genes (gyrB-rpoB-rpoD-recA), ribosomal MLSA, average nucleotide identity (ANI), in-slico DNA-DNA hybridization (DDH) were employed following standard taxono-genomic protocols.[23,24]