Chapter title |
Biochemical Approaches to Study LINE-1 Reverse Transcriptase Activity In Vitro.
|
---|---|
Chapter number | 22 |
Book title |
Transposons and Retrotransposons
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3372-3_22 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3370-9, 978-1-4939-3372-3
|
Authors |
Sébastien Viollet, Aurélien J. Doucet, Gaël Cristofari Ph.D., Gaël Cristofari, Viollet, Sébastien, Doucet, Aurélien J., Cristofari, Gaël |
Editors |
Jose L. Garcia-Pérez |
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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