Cellular and Molecular Basis of Human Biology
Lawrence S. Chan, William C. Tang in 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.
Science of biotechnology – Recombinant DNA technology
Ronald P. Evens in 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.
Methods in Molecular Biology
Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman in Molecular Imaging in Oncology, 2008
Some enzymes cut directly in the middle of the sequence, generating “blunt ends,” while others cleave both strands at different points, generating staggered ends called cohesives or “sticky” ends (Table 1). The off-staggered cleavage produces segments of DNA with complementary single-stranded ends that can be joined together again by DNA ligase. This enzyme catalyses formation of 3′ → 5′ phosphodiester bond between the 3′ -hydroxyl end of one restriction fragment strand and the 5′ -phosphate end of another during the time that the cohesive ends are transiently base paired. These cohesive or sticky ends are very useful for “cutting and pasting” DNA from different origin to create recombinant molecule with the aid of a DNA ligase. Therefore, a hybrid combination of two fragments is called recombinant DNA molecule.
Advances and challenges in drug design against tuberculosis: application of in silico approaches
Published in Expert Opinion on Drug Discovery, 2019
Alexey Aleksandrov, Hannu Myllykallio
DNA ligase plays a central role in DNA metabolism and catalyzes phosphodiester-bond formation in the backbones of DNA. Bacterial ligases are substantially different from eukaryotic ligases in structure and function as they use nicotinamide adenine dinucleotide as a co-factor, while eukaryotic ligases use adenosine-5ʹ-triphosphate. Korycka-Machala et al. [88] screened a library of 1,592 compounds from the National Cancer Institute Diversity Set against the catalytic center of Mtb ligA using the AutoDock 4.2 suite [89]. Out of 200 compounds selected by docking, 23 compounds were shortlisted for the experimental tests based on the burial inside the protein. Seven compounds out of 23 tested inhibited the activity of bacterial ligA. The most active in vitro compound also demonstrated antituberculosis activity in vivo with MIC50 of 15 µM.
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.
SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level
Published in mAbs, 2020
Christel Aebischer-Gumy, Pierre Moretti, Romain Ollier, Christelle Ries Fecourt, François Rousseau, Martin Bertschinger
Gel electrophoresis was performed using 1–2% agarose gels, prepared with UltraPureTM Agarose (Invitrogen) and 1X Tris Acetic Acid EDTA buffer (TAE, pH 8.3; Bio RAD) and stained with Gel Red Dye (Biotum). The bands of interests were cut out from the agarose gel and purified using the Gel and PCR clean-up kit (Macherey-Nagel). Ligations were performed with the T4 DNA ligase (New England Biolabs). Transformation were performed in One Shot® TOP 10 Competent E. coli (Life Technologies) and bacteria were grown with ampicillin or kanamycin (Sigma-Aldrich). Plasmid preparations were performed using the NucleoSpin Plasmid or NucleoBond Xtra Midi kits (Macherey Nagel) for minipreps and midipreps, respectively. Plasmid sequences were confirmed by Sanger sequencing at Fasteris (Plan-les-Ouates, Switzerland).
Related Knowledge Centers
- Directionality
- DNA
- DNA Repair
- DNA Replication
- Molecular Biology
- Nucleotide
- Phosphodiester Bond
- Recombinant DNA
- Lig4
- Complementarity