Which technique is best for determining transcription start sites precisely?
Accurate mapping of transcription start sites is essential for promoter analysis. Cap Analysis of Gene Expression specifically captures the 7-methylguanosine cap structure present at 5 prime ends of eukaryotic messenger RNAs. After reverse transcription, cap-trapped complementary DNAs are cleaved and 20-27 base tags adjacent to cap are isolated and sequenced, directly identifying start sites at single nucleotide resolution. RNA-seq maps entire transcripts but lacks focused resolution for initiation sites. Northern blot and SAGE measure size and abundance but cannot precisely define transcription start sites genome-wide, making CAGE superior.
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.