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Production of VNPs, VLPs, and Chimeras
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
Genetic engineering of VNPs refers to the manipulation of the genome, which results in modifications on the protein level. All plant viruses currently in use for nanotechnology applications have RNA genomes. The genomes have been sequenced, and the genetic information is available at the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). To perform genetic modifications, a cDNA copy of the genome is required. The cDNA is the complementary strand of the genome RNA, which can be synthesized by reverse transcription. The cDNA can then be amplified as double-stranded DNA using PCR techniques and inserted into a cloning or expression vector. At this stage, any standard cloning or mutagenesis procedure can be applied in order to introduce the desired modification. For detailed background information on cloning techniques, the reader is referred to textbooks in the fields of molecular biology and biochemistry.
Nanostructured Cellular Biomolecules and Their Transformation in Context of Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
Hybridization refers to the method in which molecules of nucleic acid (either single-stranded DNA or RNA) are bound to the complementary sequence, that is, adenine (A) pairs with thymine (T) or uracil (U) or vice versa and guanine (G) pairs with cytosine (C) or vice versa. Blotting is an important technique to study the hybridization in nucleic acid. The chemical basis for nucleic acid hybridization relies in the reversible helix-coil transition of the nucleic acid, which can associate as a double-stranded or dissociate into a single-stranded polymer based on the physicochemical condition of the surrounding such as temperature, ionic strength, and presence of denaturing agents. Disassociated single-stranded nucleic acid can anneal to complementary sequence forming homologous DNA (Edberg 1985).
Biomems
Published in Simona Badilescu, Muthukumaran Packirisamy, BioMEMS, 2016
Simona Badilescu, Muthukumaran Packirisamy
The core principle behind microarrays is the hybridization between two DNA strands. Hybridization is the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between the complementary nucleotide bases. A high number of complementary base pairs in a nucleotide sequence gives rise to stronger noncovalent bonding between the two strands. A glass slide is spotted or “arrayed” with DNA fragments, oligonucleotides that represent specific gene coding regions or RNA. Purified RNA is then fluorescently or radioactively labeled and hybridized to the slide. In some cases, hybridization is done simultaneously with a reference RNA to facilitate the comparison of data across multiple experiments.
Microbiology in Water-Miscible Metalworking Fluids
Published in Tribology Transactions, 2020
Frederick J. Passman, Peter Küenzi
Amplification and subsequent detection of nucleic sequences (DNA, mRNA, rRNA) are often done by application of PCR, a molecular technique that uses the ability of DNA polymerase to synthesize new strands of DNA complementary to the offered template strand. This technique can be used for microbial enumeration as well as for specification. Originally developed in the 1970s and 1980s, it has experienced many alterations and modifications since (194). Frequently, the invariable 16S rRNA gene (18S rRNA in eukaryotes) is targeted for identification and enumeration of OTUs. However, the technique is also useful for the detection of specific genes or the determination of transcriptional activity (enzyme-mediated process by which DNA gene codes are copied into messenger RNA) and is particularly useful for the detection of slow-growing microorganisms such as mycobacteria (195) in MWFs.
Overview of methodologies for the culturing, recovery and detection of Campylobacter
Published in International Journal of Environmental Health Research, 2023
Marcela Soto-Beltrán, Bertram G. Lee, Bianca A. Amézquita-López, Beatriz Quiñones
The development and implementation of additional sequence-based typing methods, including multilocus sequence typing (MLST), DNA microarrays and whole genome sequencing, have facilitated the detection and characterization of Campylobacter-related outbreaks and have enabled the improved differentiation of closely related isolates (Sabat et al. 2013). In particular, the MLST scheme for C. jejuni is based on the PCR amplification of seven highly conserved housekeeping genes, followed by the sequencing of the fragments and comparison of their nucleotide sequences using standard phylogenetic analysis. The advantage of MLST is that the data obtained is unambiguous and highly reproducible by using an internationally standardized nomenclature. As a high-throughput method, DNA microarrays technology has enabled the genotyping and profiling of genomic content in campylobacteria (Parker et al. 2006; Quiñones et al. 2007). This technology consists of a collection of DNA probes, attached in an orderly fashion to a solid surface, and the presence or absence of the complementary genome sequences in the tested isolate is detected after hybridization to the different probes on the array. Moreover, microarrays have allowed the detection of extra-genomic elements in C. jejuni (Parker et al. 2006), and pathogenic strains can also be simultaneously examined for their antimicrobial resistance and virulence potential (Quiñones et al. 2007, 2008; Sabat et al. 2013). One of the limitations of the microarray-based assays is that labeling of target DNA can be inconsistent and results in highly variable hybridization patterns. Another disadvantage is that DNA microarrays allows the identification of only those known probe sequences previously attached to the array, making it difficult to identify emerging strains that are highly variable. Also, this technique is unable to distinguish highly clonal strains based on single nucleotide polymorphisms (Sabat et al. 2013).