Run-off transcription

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) of a specific promoter along with its accuracy and rate of in vitro transcription.[1] [2][3] Run-off transcription can be used to quantitatively measure the effect of changing promoter regions on in vitro transcription levels,[1][2][4] 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.[1][2]

To perform a run-off transcription assay, a gene of interest, including the promoter, is cloned into a plasmid[4] 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.[1][2][4] DNA needs to be highly purified prior to running this assay.[1] [2] To initiate transcription, radiolabeled UTP, the other nucleotides, and RNA polymerase are added to the linearized DNA.[1][2] 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.[1][2] This fragment can then be separated by gel electrophoresis, alongside size standards, and autoradiographed.[1][2][4] 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).[4] The intensity of the band will indicate the amount of product mRNA produced.[4]


References

  1. 1 2 3 4 5 6 7 8 Loewenstein, P. M.; Song, C. Z.; Green, M (2007). "The use of in vitro transcription to probe regulatory functions of viral protein domains". Methods in molecular medicine. 131: 15–31. PMID 17656772.
  2. 1 2 3 4 5 6 7 8 "Run-off Transcription". Molecular Station. Retrieved April 16, 2014.
  3. ↑ 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 146301Freely accessible. PMID 8972868.
  4. 1 2 3 4 5 6 Allison, Lizabeth. "Fundamental molecular biology, chapter 11" (PDF). BlackWell Publishing. Retrieved April 18, 2014.
This article is issued from Wikipedia - version of the 8/20/2016. The text is available under the Creative Commons Attribution/Share Alike but additional terms may apply for the media files.