Which technique is best suited for determining transcription start sites with high precision?
CAGE, or Cap Analysis of Gene Expression, specifically captures the 7-methylguanosine cap at the 5′ end of eukaryotic mRNAs. By trapping capped RNAs, synthesizing cDNA, and sequencing a short 20–27 nucleotide tag from the extreme 5′ terminus, it maps transcription start sites at single-base resolution. This enables promoter identification, alternative promoter usage, and core promoter architecture analysis. RNA-seq suffers from 5′ end underrepresentation and SAGE samples internal tags, while northern blotting lacks nucleotide precision, making CAGE uniquely suited for TSS mapping.
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.