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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.
Recombinant DNA technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
For example, a poly-A tail may be added to the 3′-end of the RNA to make it analogous to eukaryotic mRNA (oligo-T is now used as primer). This reaction is catalyzed by the enzyme poly-A polymerase. The appropriate oligonucleotide primer (oligo-T for eukaryotic mRNA) is annealed with the mRNA. These primers will base pair to the 3′-end of mRNA. Reverse transcriptase extends the 3′-end of the primer using the mRNA molecule as a template. This produces an RNA—DNA hybrid molecule, the DNA strand being the cDNA. The RNA strand is digested by either RNase H or alkaline hydrolysis. This frees the single-stranded cDNA. Curiously, the 3′-end of this cDNA serves as its own primer and provides the free 3′-OH required for the synthesis of its complementary strand. Therefore, a primer is not required for this step. The complementary strand of the cDNA single strand is synthesized either by the reverse transcriptase itself or by E. coli DNA polymerase. This generates a hairpin loop in the cDNA. The hairpin loop is cleaved by a single strand specific nuclease to yield a regular DNA duplex.
DNA Structure, Sequencing, Synthesis, and Modification: Making Biology Molecular
Published in Richard J. Sundberg, The Chemical Century, 2017
DNA can also be cloned from RNA. This approach is particularly useful in obtaining the genes connected to specific proteins by targeting the corresponding m-RNA. In this case a reverse transcriptase is used to obtain the complementary DNA sequence, called cDNA. The process begins with isolation of all of the RNA from a particular cell line. The m-RNA is then separated, taking advantage that it contains a “polyadenylate tail” and therefore can be bound by a poly-T matrix. The purified collection of m-RNA molecules are then converted into the corresponding DNA by a reverse transcriptase. The c-DNA corresponds only to the protein sequence, because the non-coding portions (introns) have been removed during m-RNA formation. The c-DNA, in turn, can be converted to double stranded DNA by a DNA polymerase. Finally, the double stranded DNA is modified to add restriction sites that will permit its incorporation into the bacteriophage vector. Cloning then proceeds as described for DNA.
Recent applications of fluorescent nanodiamonds containing nitrogen-vacancy centers in biosensing
Published in Functional Diamond, 2022
Yuchen Feng, Qi Zhao, Yuxi Shi, Guanyue Gao, Jinfang Zhi
Under the background of COVID-19 pandemic in 2019, effective and rapid diagnosis and detection of emerging new virus has become an urgent need. Li et al. [84] proposed a quantum sensor for detecting COVID-19 based on FND with NV center (Figure 11(A, B)). Previous studies have proved that attached Gd3+ can increase the magnetic noise strength felt by NV spins and quench their T1 time. In this theoretical model, the surface of FND was modified by a cationic polymer to adsorb the DNA strand (c-DNA) complementary to the viral RNA, followed by binding Gd3+ complexes (DOTA-Gd3+). In the presence of viral RNA, the c-DNA-DOTA-Gd3+ pair could be separated from the FND surface due to the hybridization of c-DNA and viral RNA. This process is shown in Figure 11(C). The detachment of Gd3+ could result in a large change in the NV photoluminescence yield, thus the detection of virus was realized. Theoretical simulation results showed that the detection limit was as low as hundreds of viral RNA copies. The false negative rate (FNR) reached below 1%, which is far lower than the most advanced RT-PCR diagnostic method. In addition, the technology can be further promoted to diagnose other RNA viruses (like MERS) by using surface c-DNA specific to the target virus.
Biomolecules of Similar Charge Polarity Form Hybrid Gel
Published in Soft Materials, 2022
Pankaj Pandey, Vinod Kumar Aswal, Joachim Kohlbrecher, Himadri B. Bohidar
For the preparation of DNA-FA hybrid gels, typically 1% (w/v) stock DNA solution was made in deionized water and heated to 90° C with stirring for 60 min, which resulted in a homogeneous and optically clear solution. FA is not soluble in DMSO at room temperature, hence it was dissolved at 110° C and DNA in water solution was added to it dropwise to create the 1:1 (v/v) sol, which upon cooling to room temperature yielded the gel. The FA concentration was cFA = 1% (w/v), while the DNA content was varied from cDNA = 0.1 to 0.5% (w/v). The solution mixture was stirred for 30 min to obtain a homogeneous solution. Then, the samples were allowed to cool to room temperature which led to gelation (DNA–FA gel). Observation of a non-flowing meniscus of the nascent gel held inside a test tube when inverted indicated sol-gel phase transition. A systematic and rigorous study of this transition was undertaken using DLS technique which will be discussed later. All procedures were performed at room temperature of 25° C unless otherwise stated.
Synthesis of three new binuclear Mn complexes: characterization and DNA binding and cleavage properties
Published in Journal of Coordination Chemistry, 2019
Ying-Ying Kou, Qian Zhao, Xue-Rao Wang, Mei-Ling Li, Xiang-Hao Ren
To quantitatively compare the affinity of the complexes for CT-DNA, the variation in the electron absorption peak intensity as a function of the concentration of DNA can be described by the binding constant, Kb, of the complexes and DNA, calculated by the following equation [32]: where CDNA refers to the concentration of DNA base pairs and εa, εb, and εf represent the Aobsd/bCcomplex, molar absorptivity of the free coordination compounds. After plotting CDNA against CDNA/(εa − εf), the ratio of the slope and intercept of the straight line is equal to the binding constant Kb, as shown in Table 5. The experimental results confirmed that the complexes bind with DNA through an insertion mechanism.