Abstract
In vitro reverse transcriptase assays have been developed to monitor the presence and activity of ORF2p, an essential protein product of the LINE-1 retrotransposon (L1), in cellular fractions. We describe methods for expression and isolation of L1 ribonucleoprotein particles, and identification of ORF2p reverse transcriptase activity. Two independent methods are described: L1 element amplification protocol (LEAP) and direct L1 extension assay (DLEA). The first method involves cDNA synthesis by primer extension using dNTPs followed by a step of PCR amplification. The second method involves primer extension by incorporation of radiolabeled dTMPs followed by dot-blot or gel separation detection. Finally, we discuss the output and benefits of the two methods.
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Acknowledgements
We are grateful to John V. Moran (Univ. of Michigan, USA) and to Nicolas Gilbert (Institut de Génétique Humaine, France) for sharing plasmids. This work was supported by a joint Avenir grant from the Institut National de la Santé Et de la Recherche Medicale and the Institut National du Cancer [2009-340 to G.C.]; the European Research Council [243312 to G.C.]; and by Agence Nationale pour la Recherche [ANR-11-LABX-0028-01 to G.C.].
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Viollet, S., Doucet, A.J., Cristofari, G. (2016). Biochemical Approaches to Study LINE-1 Reverse Transcriptase Activity In Vitro. In: Garcia-Pérez, J. (eds) Transposons and Retrotransposons. Methods in Molecular Biology, vol 1400. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3372-3_22
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DOI: https://doi.org/10.1007/978-1-4939-3372-3_22
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