Run-off transcription assay is used to:
Run-off transcription assay quantitatively measures promoter-dependent transcription initiation in defined in vitro system. Linearized DNA template containing promoter and downstream truncated coding sequence is incubated with purified RNA polymerase, general transcription factors and radiolabeled NTPs. Polymerase initiates at transcription start site and synthesizes RNA until reaching DNA end where it falls off, generating transcript of predictable length. Product size on denaturing gel confirms initiation fidelity, while band intensity reflects efficiency. Assay is ideal for testing activators, repressors and promoter mutations and is distinct from degradation or cloning assays.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.