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A run-off transcription assay is an assay in molecular biology which is conducted in vitro to identify the position of the transcription start site (1 base pair upstream) of a specific promoter along with its accuracy and rate of in vitro transcription.
Run-off transcription can be used to quantitatively measure the effect of changing promoter regions on in vitro transcription levels, Because of its in vitro nature, however, this assay cannot accurately predict cell-specific gene transcription rates, unlike in vivo assays such as nuclear run-on.
To perform a run-off transcription assay, a gene of interest, including the promoter, is cloned into a plasmid. The plasmid is digested at a known restriction enzyme cut site downstream from the transcription start site such that the expected mRNA run-off product would be easily separated by gel electrophoresis.
DNA needs to be highly purified prior to running this assay. To initiate transcription, radiolabeled UTP, the other nucleotides, and RNA polymerase are added to the linearized DNA. Transcription continues until the RNA polymerase reaches the end of the DNA where it simply “runs off” the DNA template, resulting in an mRNA fragment of a defined length. This fragment can then be separated by gel electrophoresis, alongside size standards, and autoradiographed. The corresponding size of the band will represent the size of the mRNA from the restriction enzyme cut site to the transcription start site (+1). The intensity of the band will indicate the amount of mRNA produced.
Additionally, it can be used to detect whether or not transcription is carried out under certain conditions (i.e. in the presence of different chemicals).
References
- ^ Loewenstein, P. M.; Song, C. Z.; Green, M (2007). "The Use of in Vitro Transcription to Probe Regulatory Functions of Viral Protein Domains". Adenovirus Methods and Protocols. Methods in Molecular Medicine. Vol. 131. pp. 15–31. doi:10.1007/978-1-59745-277-9_2. ISBN 978-1-58829-901-7. PMID 17656772.
- ^ "Run-off Transcription". Molecular Station. Archived from the original on April 22, 2014. Retrieved April 16, 2014.
- Lelandais, C; Gutierres, S; Mathieu, C; Vedel, F; Remacle, C; Maréchal-Drouard, L; Brennicke, A; Binder, S; Chétrit, P (1996). "A promoter element active in run-off transcription controls the expression of two cistrons of nad and rps genes in Nicotiana sylvestris mitochondria". Nucleic Acids Research. 24 (23): 4798–804. doi:10.1093/nar/24.23.4798. PMC 146301. PMID 8972868.
- ^ Allison, Lizabeth. "Fundamental molecular biology, chapter 11" (PDF). BlackWell Publishing. Retrieved April 18, 2014.
- Sanchez, Alvaro; Osborne, Melisa L.; Friedman, Larry J.; Kondev, Jane; Gelles, Jeff (2011). "Mechanism of transcriptional repression at a bacterial promoter by analysis of single molecules". The EMBO Journal. 30 (19): 3940–3946. doi:10.1038/emboj.2011.273. PMC 3209775. PMID 21829165.