Avicenna J Med Biotech arij002 Avicenna Journal of Medical Biotechnology 2008-2835 2008-4625 Avicenna Research Institute ajmb20415 A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments ZarghampoorFarzanehBehzad-BehbahaniAbbasDepartment of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IranAzarpiraNegarResearch and Development Department, CinnaGen, Simindasht Industrial Area, Karaj, IranKhatamiSaeed RezaFanianMaryamHossein-AghdaieMahdokhtRafiei DehbidiGholam RezaDepartment of Immunology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 12 1 37 43 12 1 2019 4 5 2019

<p>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.</p> <p>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 <em>in vitro</em> 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.</p> <p>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.</p> <p>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.</p>