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

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Zarghampoor Farzaneh; BEHZAD BEHBAHANI ABBAS; AZARPIRA NEGAR; Khatami Saeed Reza; FANIAN MARYAM; HOSSEIN AGHDAIE MAHDOKHT; RAFIEI DEHBIDI GHOLAMREZA

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Journal Paper

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Journal: Avicenna Journal of Medical Biotechnology Year:2020 | Volume:12 | Issue:1 Page(s): 37-43

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Title

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

Author(s)

Keywords

Beta-globins

Mutagenesis

Polymerase chain reaction

Site-directed

Untranslated regions

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.

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References

Use of megaprimer and overlapping extension PCR (OE-PCR) to mutagenize and enhance cyclodextrin glucosyltransferase (CGTase) function

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APA: Copy

Zarghampoor, Farzaneh, BEHZAD BEHBAHANI, ABBAS, AZARPIRA, NEGAR, Khatami, Saeed Reza, FANIAN, MARYAM, HOSSEIN AGHDAIE, MAHDOKHT, & RAFIEI DEHBIDI, GHOLAMREZA. (2020). A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments. AVICENNA JOURNAL OF MEDICAL BIOTECHNOLOGY (AJMB), 12(1), 37-43. SID. https://sid.ir/paper/314060/en

Vancouver: Copy

Zarghampoor Farzaneh, BEHZAD BEHBAHANI ABBAS, AZARPIRA NEGAR, Khatami Saeed Reza, FANIAN MARYAM, HOSSEIN AGHDAIE MAHDOKHT, RAFIEI DEHBIDI GHOLAMREZA. A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments. AVICENNA JOURNAL OF MEDICAL BIOTECHNOLOGY (AJMB)[Internet]. 2020;12(1):37-43. Available from: https://sid.ir/paper/314060/en

IEEE: Copy

Farzaneh Zarghampoor, ABBAS BEHZAD BEHBAHANI, NEGAR AZARPIRA, Saeed Reza Khatami, MARYAM FANIAN, MAHDOKHT HOSSEIN AGHDAIE, and GHOLAMREZA RAFIEI DEHBIDI, “A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments,” AVICENNA JOURNAL OF MEDICAL BIOTECHNOLOGY (AJMB), vol. 12, no. 1, pp. 37–43, 2020, [Online]. Available: https://sid.ir/paper/314060/en

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