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Science of biotechnology – Recombinant DNA technology
Published in Ronald P. Evens, Biotechnology, 2020
The circular plasmid DNA must be cut open to accept the human DNA (gene) using unique bacterial enzymes (restriction endonucleases). Each endonuclease enzyme is highly specific to a certain nucleic acid sequence, creating a very specific cut, that is, an opening in the DNA plasmid structure appropriate for a specific gene’s incorporation and permitting efficient recombination. DNA materials will recombine naturally with the human gene sequence inserted into the circular plasmid sequence. A DNA ligase enzyme is employed to enhance the DNA recombination process. Figure 2.9 displays such restriction endonuclease enzymes found in nature in specific bacteria as noted. The very high specificity to an individual DNA sequence of nucleotides for endonucleases is shown in Figure 2.9.
Cellular and Molecular Basis of Human Biology
Published in Lawrence S. Chan, William C. Tang, Engineering-Medicine, 2019
These small cellular chemicals are responsible for either building up or breaking down cellular components, like DNA, RNA, or proteins. Enzymes catalyze cellular and extracellular reactions either to construct a molecule or to degrade a molecule and they make the chemical reactions go quicker than without the enzymes. They have important role of maintain a balance between providing sufficient components and eliminating excessive components in human body functions in a homeostatic way. Enzymes are generally named according to their functions. For example, the function of RNA polymerase is to synthesize RNA polymer (or RNA strand). DNA ligase functions to joint two pieces of DNA together. The six major groups of human enzymes classified by an international enzyme committee and their functional mechanisms are depicted in Table 3.
A Survey of Newer Gene Probing Techniques
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
The OLA technique requires knowledge of the target sequence. Two synthetic oligonucleotides are joined head-to-tail over the target sequence by the enzyme DNA ligase. If a mismatch occurs due to alteration (mutation) of nucleotides in the target sequence at the junction region, the ligation fails to occur.
CRISPR-based biosensing systems: a way to rapidly diagnose COVID-19
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Majid Vatankhah, Amir Azizi, Anahita Sanajouyan Langeroudi, Sajad Ataei Azimi, Imaneh Khorsand, Mohammad Amin Kerachian, Jamshid Motaei
CARP (Cas9/sgRNAs-associated reverse PCR) has been developed using the high specificity of Cas9 endonuclease and the high sensitivity of the PCR method [78]. CARP identifies target DNA in three stages. In the cleavage stage, the target DNA is cut simultaneously on two sites by Cas9 after identification of specific sequences with a pair of sgRNAs. In the ligation stage, the T4 DNA ligase ligates the cleaved DNA into intermolecular concatenated linear DNA or intramolecular circular DNA. The PCR stage is the proliferation of target DNA with a pair of reverse primers in traditional PCR or qPCR. Due to the reverse orientation of primers, target DNA amplification occurs after the cleavage and ligation stages. The CARP platform identified HPV16 and HPV18 viruses from other HPV subtypes [78]. ctPCR3.0 (CRISPR or Cas9-sgRNA typing PCR version 3) is a combination of Cas9, a pair of sgRNAs, a qPCR technique, and an isothermal incubation stage before qPCR that successfully detects target DNA in 2 h. ctPCR3.0 was able to detect the L1 and E6-E7 genes of HPV16 and HPV18 in clinical samples [79]. The LoD in the CARP and ctPCR3.0 platforms was 2 pg and 1.8 × 102 copies of target DNA, respectively [78,79]. These studies show that Cas9-based biosensing systems, as rapid, sensitive and cost-effective methods, have a high potential for virus detection.
Response of breast cancer carcinoma spheroids to combination therapy with radiation and DNA-PK inhibitor: growth arrest without a change in α/β ratio
Published in International Journal of Radiation Biology, 2020
Jing Yu, Ryan Lu, Jessie R. Nedrow, George Sgouros
DNA double-strand breaks (DSBs) caused by XRT lead to chromosomal translocations, genomic instability, and cell death if not repaired (Povirk 2006). The non-homologous end-joining (NHEJ) cell pathway is one of two well-characterized DSB repair pathways. Several core factors, including KU70/80 heterodimer and DNA-PKcs for detecting and tethering DSBs, enzymes such as Artemis and DNA polymerases for processing DNA ends, DNA ligase IV and XRCC4 complex for ligating DNA ends are involved in this pathway (Mahaney et al. 2009; Vignard et al. 2013). DNA-PKcs inhibition decreases DNA repair and increases cells’ radio-sensitivity (O’Connor et al. 2007). NU7441 is a highly potent and selective DNA-PKcs inhibitor which has been shown to sensitize many types of cancers (Ciszewski et al. 2014; Dong et al. 2018; Geng et al. 2019).
Misconnecting the dots: altered mitochondrial protein-protein interactions and their role in neurodegenerative disorders
Published in Expert Review of Proteomics, 2020
Mara Zilocchi, Mohamed Taha Moutaoufik, Matthew Jessulat, Sadhna Phanse, Khaled A. Aly, Mohan Babu
Mt biogenesis and mitophagy are two additional mt endeavors that control mt mass and functions, and alterations of this process are also involved in NDs [3,11,23]. First, mt biogenesis implies the division of preexisting organelles through the classical trio of mtDNA replication, transcription and translation. This process accommodates increased energy demands in various brain tissues, and reports show plummeting mt biogenesis with age [98]. The DNA polymerase γ (POLG) acts together with other replisome components, such as TWINKLE mtDNA helicase (also known as PEO1), mt single-stranded DNA-binding protein (mtSSB) and mt DNA ligase III to initiate the replication of mtDNA [99]. ER-mt contact sites that are spatially linked to a subset of nucleoids and marked selectively by mtDNA polymerase are engaged with mtDNA replication and division to distribute newly replicated mtDNA in human cells, suggesting a functional interdependence between mt-ER dynamics and mt genome maintenance [79].