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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]
Usually, each member of this DNA library has a T7 RNA polymerase transcription site and a ribosomal binding site at the 5’ end. The T7 promoter region allows large-scale in vitro T7 transcription to transcribe the DNA library into an mRNA library, which provides templates for the in vitro translation reaction later.
RNA polymerase, assisted by one or more general transcription factors, then selects a transcription start site in the transcription bubble, binds to an initiating NTP and an extending NTP (or a short RNA primer and an extending NTP) complementary to the transcription start site sequence, and catalyzes bond formation to yield an initial RNA product.
Abortive initiation is a normal process of transcription and occurs both in vitro and in vivo. [2] After each nucleotide-addition step in initial transcription, RNA polymerase, stochastically, can proceed on the pathway toward promoter escape (productive initiation) or can release the RNA product and revert to the RNA polymerase-promoter open complex (abortive initiation).
The G-less cassette transcription assay is a method used in molecular biology to determine promoter strength in vitro. The technique involves quantification of an mRNA product with the use of a plasmid. [1] The G-less cassette is part of a pre-constructed vector, usually containing a multiple cloning site (MCS) upstream of the cassette. For ...
A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. [1] RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
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