ArticleAvicenna journal of medical biotechnology
A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments.
Article in Avicenna journal of medical biotechnology. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed, 6 citations in OpenAlex.
- Epitope mapping via in vitro deep mutational scanning methods and its applications.The Journal of biological chemistry · 2025Review
- Establishment and evaluation of an overlap extension polymerase chain reaction technique for rapid and efficient detection of drug-resistance in Mycobacterium tuberculosis.Infectious diseases of poverty · 2022Article
- The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases.Interdisciplinary perspectives on infectious diseases · 2022Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundDespite 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.
methodsThe 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
resultsChimericGFPd2/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.
conclusionSingle 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.
Indexed as
Identifiers
32153737PMC7035467W3001444326What OpenQuestion holds
Registered trials
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