A run-off transcription assay is an assay in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
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
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
.
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
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
cut site downstream from the transcription start site such that the expected
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the ...
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
A radioactive tracer, radiotracer, or radioactive label is a chemical compound in which one or more atoms have been replaced by a radionuclide so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by tr ...
UTP, the other
nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecule ...
s, 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
{{reflist
Molecular biology techniques