Abstract
While conducting our research on mutations in the human blood platelet glycoprotein Ib-alpha (GPIbα) gene, we detected an unusual deletion of 84 bp. This deletion took place in vitro, during PCR and between two direct repeats. It was observed that the deletion could be detected either by the direct sequencing of the PCR product or after the latter’s cloning into a plasmid. After observing a series of four sequenced clones from the same individual, we noticed that while three had the same 84-bp deletion, the fourth exhibited a shorter one. We also noted that there were no cases wherein both deleted and undeleted amplicons coexisted and that several point mutations occurred in the sequence surrounding the deletion. Such Taq errors are statistically more frequent in the “deletion prone DNA” than usual. Interestingly, the deletion was observed only in a DNA, which we call here “deletion prone DNA”, whose structure might have been particularly reorganized. Indeed, the mung bean nuclease pre-treatment of this DNA prior to PCR prevented the deletion, thus strengthening the hypothesis that an intra-strand hairpin structure was involved in the deletion process. Direct repeats-mediated deletion is well known in vivo but this is the first report of such “in vitro direct repeats deletion”.
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Acknowledgements
This work is dedicated to the memory of our colleague Amira Romdhani. We would like to thank Dr. Henda Elleuch for referring us to the patient and Dr. Hayet Sellami-Kamoun for the gift of one DNA extraction kit. We are also indebted to Dr. Faiza Derbel for carefully reviewing the manuscript. We would like to express our gratitude to Dr. Ikram Guizani and Dr. Khmaies BenHaj for their help in the improvement of the manuscript. We also extend our thanks to Mr. Anouar Smaoui for carefully proofreading the language of this article.
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Hadj Kacem, B., Gargouri, J. & Gargouri, A. In Vitro Direct Repeats-mediated Deletion During PCR Amplification. Mol Biotechnol 40, 39–45 (2008). https://doi.org/10.1007/s12033-008-9059-2
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DOI: https://doi.org/10.1007/s12033-008-9059-2