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Genome Editing Tools
Published in Vineet Kumar, Vinod Kumar Garg, Sunil Kumar, Jayanta Kumar Biswas, Omics for Environmental Engineering and Microbiology Systems, 2023
Madhumita Barooah, Dibya Jyoti Hazarika
The accessibility to giant repositories associated with whole-genome sequencing as well as the understanding of previously decoded natural metabolic pathways allows redesigning of pathways through comparison with previously elucidated metabolic networks for remediation of toxic contaminants. Enzymes produced by different organisms are identified, and genes encoding those enzymes are assembled to construct novel metabolic pathways. Databases such as BRENDA (Placzek et al., 2017), KEGG (Kanehisa et al., 2017), MetaCyc (Caspi et al., 2016), and Rhea (Morgat et al., 2015) provide the information regarding the necessary enzymes for redesigning these pathways. Thus, already available pathways can be improved with additional enzymatic reactions for filling the gaps. These collective pathways are also called reference pathways and are very helpful for the comparison of various metabolic models of different organisms. BLAST (Altschul et al., 1990) – the sequence alignment program – enables the comparison of sequences by providing significant statistical similarities between the query sequences (nucleotide or protein) and the target database sequences. This approach identifies enzymes based on the fact that proteins with higher sequence homology are likely to perform similar functions.
Radiogenomics
Published in Jun Deng, Lei Xing, Big Data in Radiation Oncology, 2019
Barry S. Rosenstein, Gaurav Pandey, Corey W. Speers, Jung Hun Oh, Catharine M.L. West, Charles S. Mayo
Thus, several computational approaches have been developed to narrow the space of possible hypotheses about potential protein function, followed by experimental/literature-based validation, thus expediting the overall process. The first natural approach was to use sequence homology assessment tools, such as the Basic Local Alignment Search Tool (BLAST) and Position-Specific Iterative BLAST (PSI-BLAST) (Altschul et al. 1990, 1997), to transfer functional annotations to unannotated proteins from proteins having similar amino acid sequences. However, other studies demonstrated that this approach does not always yield accurate results due to the multidomain structure of proteins and the insufficiency of sequence homology to reflect the effects of the evolutionary process of gene duplication (Gerlt and Babbitt 2000; Whisstock and Lesk 2003). Thus, a much wider spectrum of data types was leveraged to expand the types, specificity, and accuracy of protein functions that can be predicted. Appropriate data analysis methods, including those from machine learning (e.g., clustering, classification, and network analysis), were employed to infer protein function form these data types. Table 13.1 lists the most well-investigated data types, as well as the most prominent data analysis approaches, used to predict protein function. For details of these data types and approaches, we refer the reader to extensive reviews (Pandey et al. 2006; Lee et al. 2007; Sharan et al. 2007) and reports of recent large-scale assessments (Radivojac et al. 2013; Jiang et al. 2016).
Principles and Techniques for Deoxyribonucleic Acid (DNA) Manipulation
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
Nwadiuto (Diuto) Esiobu, Ifeoma M. Ezeonu, Francisca Nwaokorie
Basic research – A comparison of sequence homology of conserved genes in different organisms can be made. It is even possible to study extinct organisms using samples of material from bones or museum specimens. The DNA used for PCR amplification can be partially degraded. As long as a few DNA molecules are intact between the two primers, amplification can occur.
The application of molecular tools to study the drinking water microbiome – Current understanding and future needs
Published in Critical Reviews in Environmental Science and Technology, 2019
The second type of molecular method is to obtain a dataset of 16S rRNA gene sequences from the extracted community genomic DNA. Initially, this was achieved through clone library construction of 16S rRNA gene sequences. In the recent years, the composition of 16S rRNA gene sequences in a microbial sample can be obtained using the next-generation sequencing (NGS) technology. Both approaches describe the microbial composition based on the number of unique 16S rRNA sequences and the abundance of individual 16S rRNA sequences. The 16S rRNA sequences can be further compared with all 16S rRNA sequences stored in a public database. This allows one to infer the phylogeny affiliation of individual 16S rRNA sequences, and determine whether the sequences are novel or related to known organisms based on the similarity of sequence homology (e.g., <97% similarity for defining a new species). Furthermore, based on the sequence information, one can design oligonucleotide probes specific for a target organism or a group of organisms, and then apply them in whole-cell hybridization or membrane hybridization for confirming the presence of the targeted organisms or for quantitative measurement of those targeted organisms in the environment. Likewise, the active members within the microbial community can be determined using the corresponding RNA-based analysis instead of DNA-based methods.
Optimization of cultivation strategy and medium for bacteriocin activity of Enterococcus faecium HDX-2
Published in Preparative Biochemistry & Biotechnology, 2021
Renpeng Du, Fangyi Pei, Jie Kang, Wen Zhang, Wenxiang Ping, Hongzhi Ling, Jingping Ge
The 16S rDNA sequence analysis technology was used to further identify the strain. The general primers for PCR were as follows: 8 F: 5′–AGAGTTTGATCATGGCTCAG-3′ and 1492 R: 5′–ACGGTTACCTTGTTACGACTT-3′. PCR conditions included a first denaturing steep for 3 min at 95 °C, followed by 30 cycles: denaturing for 30 s at 95 °C, annealing for 60 s at 55 °C and extension for 90 s at 72 °C and finally extension for 5 min at 72 °C.[17] GENEWIZ company (Beijing, China) was commissioned to sequence the PCR products. The sequence obtained was compared with the National Center for Biotechnology Information (NCBI) database to determine the sequence homology.