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

Farzaneh Zarghampoor; Abbas Behzad-Behbahani; Negar Azarpira; Saeed Reza Khatami; Maryam Fanian; Mahdokht Hossein Aghdaie; Gholamreza Rafiei Dehbidi

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

Avicenna J Med Biotechnol. 2020 Jan-Mar;12(1):37-43.

Authors

Farzaneh Zarghampoor¹, Abbas Behzad-Behbahani², Negar Azarpira³, Saeed Reza Khatami¹, Maryam Fanian³, Mahdokht Hossein Aghdaie³, Gholamreza Rafiei Dehbidi²

Affiliations

¹ Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
² Diagnostic Laboratory Sciences and Technology Research Centre, Faculty of Paramedical, Shiraz Sciences University of Medical Sciences, Shiraz, Iran.
³ Transplant Research Centre, Shiraz University of Medical Science, Shiraz, Iran.

PMID: 32153737
PMCID: PMC7035467

Abstract

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.

Keywords: Beta-globins; Mutagenesis; Polymerase chain reaction; Site-directed; Untranslated regions.