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.

http://www.biotechrep.ir/article_100740_fa1add16041b371144b3e24b68c454b0.pdf

Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

Recombination is a common evolutionary tool for RNA viruses, and coronaviruses are no exception. We review here the evidence for recombination in SARS-CoV-2 and reconcile nomenclature for recombinants, discuss their origin and fitness, and speculate how recombinants could make a difference in the future of the COVID-19 pandemics.

/pdf/recombination-in-coronaviruses-with-a-focus-on-sars-cov-2-1kn1h3n8.pdf

Recombination in Coronaviruses, with a Focus on SARS-CoV-2

(1) Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) excretion in stools is well documented by RT-PCR, but evidences that stools contain infectious particles are scarce. (2) Methods: After observing a Corona Virus 2019 Disease (COVID-19) epidemic cluster associated with a ruptured sewage pipe, we search for such a viable SARS-CoV-2 particle in stool by inoculating 106 samples from 46 patients. (3) Results: We successfully obtained two isolates from a unique patient with kidney transplantation under immunosuppressive therapy who was admitted for severe diarrhea. (4) Conclusions: This report emphasizes that SARS-CoV-2 is an enteric virus, and infectious virus particles can be isolated from the stool of immune-compromised patients like, in our case, kidney transplant recipient. Immune-compromised patients are likely to have massive multiplication of the virus in the gastrointestinal tract and this report suggests possible fecal transmission of SARS-CoV-2.

https://hal-amu.archives-ouvertes.fr/hal-03333886/document

Isolation of Viable SARS-CoV-2 Virus from Feces of an Immunocompromised Patient Suggesting a Possible Fecal Mode of Transmission.

Highly deleterious variations in COX1, CYTB, SCG5, FK2, PRL and PGF genes are the potential adaptation of the immigrated African ostrich population

Article history: Received April 19, 2020 Accepted May 14, 2020 Available online June 23, 2021 This study was conducted to determine the genetic variation of the prolactin (PRL) gene in 5' flanking region and its role with several reproductive traits in Awassi sheep. Onehundred six Iraqi sexually mature and healthy Awassi ewes that aged between 2 and 2.5 years were included in this study. Ewes were classified into two the main divisions: ewes producing twins and ewes producing single offspring. Two genotypes (AA and AT) were observed in Awassi sheep. While genotype AA was detected in Awassi ewes that produced twins, genotype AT was detected in ewes that produced a single offspring. The sequencing reactions identified nine SNPs in the prolactin gene at the 5′ flanking region in Awassi sheep, differing from the prolactin reference sequence (GenBank accession number X16641.1). Genotype AT possessed one single nucleotide polymorphism SNP substitute comparison with the AA genotype in Awassi ewes. The association analysis revealed that the AA genotype is characterized by significantly higher levels of the progesterone concentration, twinning ratio, fecundity, and prolificacy than the AT genotype. In conclusion, a new SNP (g.1209 A>T) was discovered within the ovine flanking region which potentially influences prolactin gene expression. These results showed that the genotype AA associated with high prolificacy of Awassi sheep may be used as a selection criterion for improving the reproductive performance of Iraqi Awassi sheep.

/pdf/novel-single-nucleotide-polymorphism-in-the-prolactin-gene-969sl29y86.pdf

Novel single nucleotide polymorphism in the prolactin gene of Awassi ewes and its role in the reproductive traits

There is no 'one' procedure for extracting DNA from the whole blood of both mammals and birds, since each species has a unique property that require different methods to release its own DNA. Therefore, to obtain genomic DNA, a universal, rapid, and noncostly method was developed. A very simple biological basis is followed in this procedure, in which, when the blood is placed in water, it rapidly enters the RBCs by osmosis and causes cells to burst by hemolysis. The validity of extracting genomic DNA was confirmed by several molecular biological experiments. It was found that this method provides an efficient and versatile alternative for extracting bulk amounts of highly-qualified DNA from the blood of a wide range of species. This is the first manuscript that describes use of distilled water as the only eliminator of RBCs among all other known DNA extraction techniques.

A universal, rapid, and inexpensive method for genomic DNA isolation from the whole blood of mammals and birds.

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.

/pdf/infection-with-different-strains-of-sars-cov-2-in-patients-2gi48f65ye.pdf

Infection with different strains of SARS-CoV-2 in patients with COVID-19

GREM1 (gremlin1) is a known inhibitor for BMP15 (bone morphogenetic protein 15) family, but its genetic diversity in sheep is unknown. The present study was conducted to analyze the polymorphism of GREM1 gene using PCR- single-strand conformation polymorphism (SSCP) and DNA sequencing methods and to assess the possible association of GREM1 gene polymorphism with reproductive traits in Awassi ewes. A total of 224 ewes, 124 producing singles and 100 producing twins, were included in the study. Two SSCP patterns were detected in two amplified loci within the exon 2. Two exonic novel single nucleotide polymorphism (SNP)s were identified, c.74 T > G (the silent SNP p.Met123 =) and c.30 T > A with (the missense SNP p.Ile237Phe). Statistical analyses indicated a non-significant (P > 0.05) association of p.Met123 = with the analyzed reproductive traits of fecundity, prolificacy, litter size, and twinning rate. Meanwhile, p.Ile237Phe SNP exhibited a highly significant (P < 0.01) association with the measured reproductive traits, in which ewes with TA genotype (with p.Ile237Phe SNP) exhibited higher litter size, twinning ratio, fecundity, and prolificacy than those with TT genotype (without p.Ile237Phe SNP). The deleterious impact of p.Ile237Phe SNP was observed by the means of ten different state-of-the-art in silico tools that predicted a highly damaging effect of p.Ile237Phe SNP on the structure, function, and stability of gremlin1. In conclusion, the results of our study suggest that p.Ile237Phe SNP has a remarkable negative impact on the gremlin1 structure, function, and stability. Since gremlin1 is a known inhibitor of reproductive performance, a consequent higher reproductive performance was observed in ewes with damaged gremlin1 (with p.Ile237Phe SNP) than those with non-damaged gremlin1 (without p.Ile237Phe SNP). Therefore, it can be stated that the implementation of the novel p.Ile237Phe SNP in the GREM1 gene could be a useful marker in marker-assisted selection. This manuscript is the first one to describe GREM1 gene variations in sheep.

Mohammed Baqur S. Al-Shuhaib

Papers

Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

A universal, rapid, and inexpensive method for genomic DNA isolation from the whole blood of mammals and birds.

Highly deleterious variations in COX1, CYTB, SCG5, FK2, PRL and PGF genes are the potential adaptation of the immigrated African ostrich population

Infection with different strains of SARS-CoV-2 in patients with COVID-19

A Novel Missense Single Nucleotide Polymorphism in the GREM1 Gene is Highly Associated with Higher Reproductive Traits in Awassi Sheep