A run-off transcription assay is an assay in
molecular biology
Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
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
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...
.
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 Protein biosynthesis, synthesizing a protein.
mRNA is ...
run-off product would be easily separated by
gel electrophoresis
Gel electrophoresis is an electrophoresis method for separation and analysis of biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge through a gel. It is used in clinical chemistry to separate ...
.
DNA needs to be highly purified prior to running this assay.
To initiate transcription,
radiolabeled UTP, the other
nucleotide
Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...
s, and
RNA polymerase
In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template.
Using the e ...
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
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Molecular biology techniques