Biochemical Approaches to Study LINE-1 Reverse Transcriptase Activity In Vitro

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.