title: Biochemical Approaches to Study LINE-1 Reverse Transcriptase Activity In Vitro.
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.
authors.affiliation: INSERM, U1081, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, 06100, France.|CNRS, UMR 7284, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, 06100, France.|Faculty of Medicine, University of Nice-Sophia-Antipolis, Nice, 06100, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Viollet
authors.affiliation: INSERM, U1081, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, 06100, France.|CNRS, UMR 7284, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, 06100, France.|Faculty of Medicine, University of Nice-Sophia-Antipolis, Nice, 06100, France.
authors.forename: Aurélien J
authors.initials: AJ
authors.lastname: Doucet
authors.affiliation: INSERM, U1081, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, 06100, France. Gael.Cristofari@unice.fr.|CNRS, UMR 7284, Institute for Research on Cancer and Aging of Nice (IRCAN), Nice, 06100, France. Gael.Cristofari@unice.fr.|Faculty of Medicine, University of Nice-Sophia-Antipolis, Nice, 06100, France. Gael.Cristofari@unice.fr.
authors.forename: Gaël
authors.initials: G
authors.lastname: Cristofari
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D046912:Multiprotein Complexes; D016366:Open Reading Frames; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D012261:Ribonucleoproteins; D012194:RNA-Directed DNA Polymerase
keywords: In vitro assay; L1; LINE-1; Non-LTR retrotransposon; ORF2p; RNA-dependent DNA-polymerase; Retroelement; Reverse transcriptase; Ribonucleoprotein particle