A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments.

BACKGROUND: Despite the ease of conventional splicing by overlap-extension (SOEing) PCR technique in theory, when splicing more than two fragments, and especially if one of the complementary sequences is A-T rich, the attachment of the fragments would be challenging. A new rapid and highly efficient SOEing PCR assay was developed for simultaneous splicing of multiple DNA fragments and induction of site-directed mutagenesis in a single tube. METHODS: The method was adapted for splicing human beta-globin UTRs to OCT4, SOX2, KLF4, C-MYC, LIN28A, and destabilized GFP for the construction of chimeric DNA fragments for in vitro transcription. In addition, the native Kozak sequence of beta-globin (K1) was replaced by the strongest Kozak sequence (K2) using site-directed mutagenesis to enhance the expression of target genes. RESULTS: ChimericGFPd2/K1, GFPd2/K2, OCT4, and KLF4 were created by the optimized conventional SOEing PCR. The single tube method was able to create the chimeric SOX2, C-MYC, and LIN28A in high quality and quantity in comparison with the conventional SOEing PCR. Moreover, using single tube SOEing PCR, the reaction time and materials that are required in the conventional SOEing PCR were significantly reduced. Fluorescent microscopy and flow cytometry examinations indicated highly efficient translation of K2 sequence in comparison with the K1sequence. CONCLUSION: Single tube SOEing PCR is a valuable method to construct more multiple fragments with high yield. The method can successfully be applied for construction of various kinds of complex chimeric genes.

Improved translation efficiency of therapeutic mRNA.

Recent developments in the field of the messenger RNA and its advantages versus DNA have led to a renewed interest in mRNA-based technologies. Despite its advantages, mRNA therapy has a number of drawbacks including low amount of mRNA production, short-term existence of mRNA and mRNA-mediated protein within the cell, severe mRNA cytotoxicity, and immune response activation following mRNA transfection. Here, we applied untranslated regions of human beta-globin to increase the stability and translation efficiency of a destabilized GFP mRNA. In order to suppress the innate immune response, which is the main barrier of mRNA therapy, we used the vaccinia virus derived capping enzyme and substituted standard nucleotides with modified nucleotides. At the end, the Kozak sequence of human beta-globin was replaced with the strongest sequence for the further improvement of mRNA translation. Overall, these modifications with native Kozak (K1) sequence of human beta-globin enhanced the stability of destabilized GFP mRNA up to 48 h and no increase in the level of interferon-α and -ß was found. The GFP expression of mRNA with modified Kozak (K2) sequence initiated earlier than mRNA and plasmid DNA with K1 sequence. In contrast to mRNA with K1 sequence, the cells containing mRNA with K2 sequence remained positive for GFP expression up to 72 h post-transfection. Interestingly, transfection efficiency and mean fluorescence intensity (MFI) of mRNA with K2 sequence were higher than mRNA and plasmid DNA with K1 sequence. Taken together, these results provide valuable information for the optimization of mRNA stability and translation. Therefore, the methods used in the current study can successfully be applied for reprogramming, gene editing, trans-differentiation, tumour therapy, and gene therapy.

A Panel of Cancer Testis Antigens and Clinical Risk Factors to Predict Metastasis in Colorectal Cancer.

Colorectal cancer (CRC) is the third common carcinoma with a high rate of mortality worldwide and several studies have investigated some molecular and clinicopathological markers for diagnosis and prognosis of its malignant phenotypes. The aim of this study is to evaluate expression frequency of PAGE4, SCP-1, and SPANXA/D cancer testis antigen (CTA) genes as well as some clinical risk markers to predict liver metastasis of colorectal cancer patients. The expression frequency of PAGE4, SCP-1, and SPANXA/D cancer/testis antigen (CTA) genes was obtained using reverse transcription polymerase chain reaction (RT-PCR) assay in 90 colorectal tumor samples including both negative and positive liver metastasis tumors. Statistical analysis was performed to assess the association of three studied genes and clinical risk factors with CRC liver metastasis. The frequency of PAGE4 and SCP-1 genes expression was significantly higher in the primary tumours with liver metastasis when statistically compared with primary tumors with no liver metastasis (P < 0.05). Among all clinical risk factors studied, the lymph node metastasis and the depth of invasion were statistically correlated with liver metastasis of CRC patients. In addition, using multiple logistic regression, we constructed a model based on PAGE4 and lymph node metastasis to predict liver metastasis of CRC.

Lack of association between the G-660C polymorphism in the dopamine transporter gene (SLC6A3) and schizophrenia in the Iranian population.

BACKGROUND: Dopaminergenic system plays an essential role in the plasticity of the human brain. The dopamine transporter gene (SLC6A3) mediates active reuptake of dopamine from synapsis, terminates dopamine signals, and therefore, is implicated in a number of dopamine-related disorders like psychosis. Variations in the form of single nucleotide polymorphisms in the core promoter of the SLC6A3 gene are reported to be involved in the pathogenesis of schizophrenia. In this study, we also attempted to establish the possible role of the polymorphism G-660C in the SLC6A3 gene promoter in schizophrenia in a case-control study. MATERIALS AND METHODS: The allele and genotype frequency were analyzed in an Iranian cohort of 200 unrelated patients and 200 controls using polymerase chain reaction and restriction fragment length polymorphism. RESULTS: The genotype frequency for case and control groups was GG 100%, GC 0%, CC 0%, and GG 100%, GC 0%, CC 0%, respectively. The C allele was failed in both groups. CONCLUSION: Our data suggest clearly that there is no association between the -660G/C polymorphism and outcome of schizophrenia in the Iranian population.