Zarghampoor Farzaneh, Behzad-Behbahani Abbas, Azarpira Negar, Khatami Saeed Reza, Fanian Maryam, Hossein Aghdaie Mahdokht, Rafiei Dehbidi Gholamreza
Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Diagnostic Laboratory Sciences and Technology Research Centre, Faculty of Paramedical, Shiraz Sciences University of Medical Sciences, Shiraz, Iran.
Avicenna J Med Biotechnol. 2020 Jan-Mar;12(1):37-43.
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.
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 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.
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.
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.
尽管理论上常规的重叠延伸拼接（SOEing）PCR技术操作简便，但在拼接两个以上片段时，尤其是当互补序列之一富含A-T时，片段的连接会具有挑战性。我们开发了一种新的快速高效的SOEing PCR检测方法，可在单管中同时拼接多个DNA片段并诱导定点诱变。
该方法适用于将人β-珠蛋白非翻译区（UTR）与OCT4、SOX2、KLF4、C-MYC、LIN28A和不稳定绿色荧光蛋白（GFP）进行拼接，以构建用于转录的嵌合DNA片段。此外，利用定点诱变将β-珠蛋白的天然科扎克序列（K1）替换为最强的科扎克序列（K2），以增强靶基因的表达。
通过优化的常规SOEing PCR构建了嵌合GFPd2/K1、GFPd2/K2、OCT4和KLF4。与传统的SOEing PCR相比，单管法能够高质量、高产量地构建嵌合SOX2、C-MYC和LIN28A。此外，使用单管SOEing PCR，传统SOEing PCR所需的反应时间和材料显著减少。荧光显微镜和流式细胞术检查表明，与K1序列相比，K2序列的翻译效率更高。
单管SOEing PCR是一种构建多个片段且产量高的有价值方法。该方法可成功应用于构建各种复杂的嵌合基因。
