LEAP: L1 Element Amplification Protocol.

Autor:	Kopera HC; Department of Human Genetics, University of Michigan Medical School, 1241 E. Catherine Street, Ann Arbor, MI, 48109, USA. chongh@umich.edu., Flasch DA; Department of Human Genetics, University of Michigan Medical School, 1241 E. Catherine Street, Ann Arbor, MI, 48109, USA., Nakamura M; Department of Human Genetics, University of Michigan Medical School, 1241 E. Catherine Street, Ann Arbor, MI, 48109, USA., Miyoshi T; Department of Human Genetics, University of Michigan Medical School, 1241 E. Catherine Street, Ann Arbor, MI, 48109, USA., Doucet AJ; Department of Human Genetics, University of Michigan Medical School, 1241 E. Catherine Street, Ann Arbor, MI, 48109, USA., Moran JV; Department of Human Genetics, University of Michigan Medical School, 1241 E. Catherine Street, Ann Arbor, MI, 48109, USA. moranj@umich.edu.; Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, MI, USA. moranj@umich.edu.; Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA. moranj@umich.edu.; Howard Hughes Medical Institute, Chevy Chase, MD, USA. moranj@umich.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2016; Vol. 1400, pp. 339-55.
DOI:	10.1007/978-1-4939-3372-3_21
Abstrakt:	Long INterspersed Element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that are required for retrotransposition. The L1 element amplification protocol (LEAP) assays the ability of L1 ORF2p to reverse transcribe L1 RNA in vitro. Ultracentrifugation or immunoprecipitation is used to isolate L1 ribonucleoprotein particle (RNP) complexes from cultured human cells transfected with an engineered L1 expression construct. The isolated RNPs are incubated with an oligonucleotide that contains a unique sequence at its 5' end and a thymidine-rich sequence at its 3' end. The addition of dNTPs to the reaction allows L1 ORF2p bound to L1 RNA to generate L1 cDNA. The resultant L1 cDNAs then are amplified using polymerase chain reaction (PCR) and the products are visualized by gel electrophoresis. Sequencing the resultant PCR products then allows product verification. The LEAP assay has been instrumental in determining how mutations in L1 ORF1p and ORF2p affect L1 reverse transcriptase (RT) activity. Furthermore, the LEAP assay has revealed that the L1 ORF2p RT can extend a DNA primer with mismatched 3' terminal bases when it is annealed to an L1 RNA template. As the LINE-1 biology field gravitates toward studying cellular proteins that regulate LINE-1, molecular genetic and biochemical approaches such as LEAP, in conjunction with the LINE-1-cultured cell retrotransposition assay, are essential to dissect the molecular mechanism of L1 retrotransposition. Competing Interests: J.V.M. is an inventor on the patent: “Kazazian, H.H., Boeke, J.D., Moran, J.V., and Dombrowski, B.A. Compositions and methods of use of mammalian retrotransposons. Application No. 60/006,831; Patent No. 6,150,160; Issued November 21, 2000.” J.V.M. has not made any money from this patent and voluntarily discloses this information.
Databáze:	MEDLINE
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