Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) are two independent methods used in the post-amplification genotyping of DNA variations. Both techniques are used in a wide range of screening applications to characterize single nucleotide polymorphisms (SNPs). The PCR-SSCP enables the identification of a potentially causative unknown SNP that could not be identified by PCR-RFLP. However, because complicated steps are not required to perform PCR-RFLP, it is used in many applications. On the other hand, PCR-RFLP is easier to process in terms of time and handover experience, the detection of a particular unknown SNP by PCR-SSCP has further chances. The simplicity of PCR-RFLP does not mean that it is better than PCR-SSCP. The reason is the limited ability of PCR-RFLP to detect nucleotide variations, which often go undetected because each restriction enzyme (RE) scans only a few recognition sequences, and other sequences are ignored. Furthermore, the efficacy of PCR-SSCP is sometimes hindered by many optimizations and also lack of experience. As PCR-SSCP allows other sequences within an amplicon to be separated and characterized, the choice between PCR-RFLP and PCR-SSCP is largely dependent on the reason for each genotyping experiment. This review provides a useful guide for comparing PCR-RFLP and PCR-SSCP in terms of their concepts, efficiency, ease of use, interpretation, and sensitivity as well as several other parameters. The comparison is extended to the practical applications of both techniques in terms of their utilization in molecular diagnostics and related applications.
The biological diversity of SARS-CoV-2 was assessed by investigating the genetic variations of the spike glycoprotein of patients with COVID-19 in Iraq. Sequencing identified fifteen novel nucleic acid variations with a variety of distributions within the investigated samples. The electropherograms of all identified variations showed obvious co-infections with two different viral strains per sample. Most samples exhibited three nonsense single nucleotide polymorphism (SNPs), p.301Cdel, p.380Ydel and p.436del, which yielded three truncated spike glycoproteins, respectively. Network and phylogenetic analyses indicated that all viral infections were derived from multiple viral origins. Results inferred from the specific clade-based tree showed that some viral strains were derived from European G-clade sequences. Our data demonstrated the absence of single-strain infection among all investigated samples in the studied area, which entails a higher risk of SARS-CoV-2 in this country. The identified high frequency of truncated spike proteins suggests that defective SARS-CoV-2 depend on helper strains possessing intact spikes during infection. Alternatively, another putative ACE2-independent route of viral infection is suggested. To the best of our knowledge, this is the first report to describe co-infection with multiple strains of SARS-CoV- 2 in patients with COVID-19.
Angiotensin-converting enzyme 2 (ACE2) is the first target of SARS-CoV-2 and a key functional host receptor through which this virus hooks into and infects human cells. The necessity to block this receptor is one of the essential means to prevent the outbreak of COVID-19. This study was conducted to determine the most eligible natural compound to suppress ACE2 to counterfeit its interaction with the viral infection. To do this, the most known compounds of sixty-six Iraqi medicinal plants were generated and retrieved from PubChem database. After preparing a library for Iraqi medicinal plants, 3663 unique ligands’ conformers were docked to ACE2 using the GLIDE tool. Results found that twenty-three compounds exhibited the highest binding affinity with ACE2. The druglikeness and toxicity potentials of these compounds were evaluated using SwissADME and Protox servers respectively. Out of these virtually screened twenty-three compounds, epicatechin and kempferol were predicted to exert the highest druglikeness and lowest toxicity potentials. Extended Molecular dynamics (MD) simulations showed that ACE2-epicatechin complex exhibited a slightly higher binding stability than ACE2-kempferol complex. In addition to the well-known ACE2 inhibitors that were identified in previous studies, this study revealed for the first time that epicatechin from Hypericum perforatum provided a better static and dynamic inhibition for ACE2 with highly favourable pharmacokinetic properties than the other known ACE2 inhibiting compounds. This study entailed the ability of epicatechin to be used as a potent natural inhibitor that can be used to block or at least weaken the SARS-CoV-2 entry and its subsequent invasion. In vitro experiments are required to validate epicatechin effectiveness against the activity of the human ACE2 receptor.
Objectives: Polyphenols are vital micronutrients, in our diet, which have a role in the prevention of progressive cancer and cardiovascular diseases developing. The main objective of this research was to evaluate the hemopreventive effects of some polyphenols of Phoenix dactylifera pits on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary cancer of female albino rats.Methods: The phenolics of P. dactylifera pits (Zahidi cultivar) were prepared by successive steps; extraction by ethanol:methanol:HCl:H2O, adsorption chromatography using a silica gel column and preparative high performers chromatography. The cytotoxic activity of the phenolics was detected against human breast cancer cell line (MCF-7). The acetone phenolic fraction, 50 female albino rats, and DMBA carcinogen were used to study the preventive effects.Results: The acetone phenolic fraction contained considerable amounts of the total phenolics and having antioxidant activity, which inhibit cancer cell line MCF-7 growth, and the inhibitory concentration 50% of cells (IC50) at 24 hrs was 202 μg/ml for MCF-7 and 480 μg/ml for WRL-68. The purified phenolic compounds may be related to genistein and quercetin derivatives that inhibited MCF-7 cell line growth and IC50 were 1030 and 203.9 μg/ml, respectively, compared with negligible effects on normal cell line. The acetone phenolic fraction prevented mammary cancer formation in the DMBA-induced rat model.Conclusions: The phenolics of date palm had chemopreventive effects against DMBA-induced mammary cancer, and they required further research to clarify the possible mechanisms that might have contributed to the preventive effects against mammary cancer.
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