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#DNA template

3 public questions tagged with this topic.

Transcript analysis

Which assay is in vitro and uses a cut DNA template to study transcription rate?

Run-off transcription assay evaluates promoter strength and accuracy of initiation in defined in vitro system. A DNA template containing promoter of interest is linearized at defined position downstream of transcription start site using restriction enzyme, creating truncated template. Addition of RNA polymerase holoenzyme with sigma factor and ribonucleoside triphosphates results in transcription initiation at promoter and elongation until enzyme runs off at cut end, producing transcript of predicted length measurable on urea-polyacrylamide gel. Amount of run-off product quantifies transcripti

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.

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Transcriptome analysis

Which in vitro assay measures the rate of transcription from a cut DNA template?

Run-off transcription is an in vitro biochemical assay designed to study promoter strength and transcription factor requirements. A DNA template containing a promoter followed by a defined sequence is linearized downstream with restriction enzymes, causing RNA polymerase to transcribe and then fall off at the cut site, producing a transcript of fixed length. Reactions include labeled UTP and nuclear extracts or purified factors. Quantity of RNA synthesized is measured by autoradiography. Northern blot detects steady-state RNA, SAGE quantifies tags, and ChIP assesses protein occupancy, not tran

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.

View Details