Background: The invention of Polymerase Chain Reaction (PCR) marked a breakthrough in biomedical research. Its invention divided the timeline into an era before and after PCR. Because of its multiple applications, it has become a vital tool for clinical researchers and diagnosticians. A effective PCR experiment requires adequate knowledge of each reaction component and stepby- step procedure to attain the optimized results. This research aims to employ optimization strategies that are easy to perform, cost-effective, and do not require PCR kits for the generation of amplicons for TYR, MITF, and SOX10 genes and can be used in sequence analysis. Materials and Methods: Whole blood samples were used to extract genomic DNA with an inorganic method. DNA quantification was done by spectrophotometry analysis. Optimization strategies were adopted to generate PCR products of candidate genes and visualized by agarose gel electrophoresis. Sanger sequencing was performed to check the quality and specificity of generated amplicons via optimization strategies. Results: This study demonstrates a novel approach for troubleshooting failed reactions without the use of PCR kits. The result indicated that use of sterilized material and reagents along with optimum MgCl2 concentration (2.0-3.0 mM), DNA quantity (25 ng/μL), and annealing temperature (54-60°C) are necessary to achieve successful amplification. Sequence analysis revealed no background noise often associated with sequencing results. Conclusion: Optimizing MgCl2 concentration, DNA quantity, annealing temperature, along with the use of contamination-free material and reagents are essential steps in PCR optimization. Following this guide, anyone lacking proper supervision, and with little or no knowledge of the procedure should attain the desired results.

Congenital idiopathic nystagmus (CIN) is an oculomotor disorder characterized by repetitive and rapid involuntary movement of the eye that usually develops in the first six months after birth. Unlike other forms of nystagmus, CIN is widely associated with mutations in the FRMD7 gene. This study involves the molecular genetic analysis of a consanguineous Pakistani family with individuals suffering from CIN to undermine any potential pathogenic mutations. Blood samples were taken from affected and normal individuals of the family. Genomic DNA was extracted using an in-organic method. Whole Exome Sequencing (WES) and analysis were performed to find any mutations in the causative gene. To validate the existence and co-segregation of the FRMD7 gene variant found using WES, sanger sequencing was also carried out using primers that targeted all of the FRMD7 coding exons. Additionally, the pathogenicity of the identified variant was assessed using different bioinformatic tools. The WES results identified a novel nonsense mutation in the FRMD7 (c.443T>A; p. Leu148 *) gene in affected individuals from the Pakistani family, with CIN resulting in a premature termination codon, further resulting in the formation of a destabilized protein structure that was incomplete. Co-segregation analysis revealed that affected males are hemizygous for the mutated allele c.443T>A; p. Leu148 * and the affected mother is heterozygous. Overall, such molecular genetic studies expand our current knowledge of the mutations associated with the FRMD7 gene in Pakistani families with CIN and significantly enhance our understanding of the molecular mechanisms involved in genetic disorders.

One of the aggressive and lethal cancers, pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and resistance to conventional treatments. Moreover, the tumor immune microenvironment (TIME) plays a crucial role in the progression and therapeutic resistance of PDAC. It is associated with T-cell exhaustion, leading to the progressive loss of T-cell functions with an impaired ability to kill tumor cells. Therefore, this study employed single-cell RNA sequencing (scRNA-seq) analysis of a publicly available human PDAC dataset, with cells isolated from the primary tumor and adjacent normal tissues, identifying upregulated genes of T-cells and cancer cells in two groups (“cancer cells_vs_all-PDAC” and “cancer-PDAC_vs_all-normal”). Common and unique markers of cancer cells from both groups were identified. The Reactome pathways of cancer and T-cells were selected, while the genes implicated in those pathways were used to perform PPI analysis, revealing the hub genes of cancer and T-cells. The gene expression validation of cancer and T-cells hub-genes was performed using GEPIA2 and TISCH2, while the overall survival analysis of cancer cells hub-genes was performed using GEPIA2. Conclusively, this study unraveled 16 novel markers of cancer and T-cells, providing the groundwork for future research into the immune landscape of PDAC, particularly T-cell exhaustion. However, further clinical studies are needed to validate these novel markers as potential therapeutic targets in PDAC patients.

Eye colour in humans is a distinct character, and an important trait of an individual’s personality. Heterochromia iridis is a unique phenotype. It is an exceedingly rare condition caused by mutations in genes responsible for synthesis, distribution, and concentration of the melanin pigment in the irises of the eye. Many health disorders and syndromes are associated with heterochromia. Here, we report for the first time in Pakistan, two unique individuals with sectoral heterochromia. This report also addresses the social and psychological pressures these remarkable individuals face in the Pakistani society.