Which molecule is added in radiolabeled form in ROTA?
In Run-Off Transcription Assay, radiolabeled nucleotide triphosphates, commonly alpha-32P-UTP or alpha-32P-ATP, are included in transcription reaction mixture. As RNA polymerase synthesizes RNA, these labeled precursors are incorporated into nascent transcript, allowing sensitive autoradiographic detection and quantification after gel separation. Use of labeled nucleotides provides direct readout of newly synthesized RNA without modifying template DNA, polymerase enzyme, or other protein factors. Labeling DNA or protein would not reflect transcriptional activity. This approach enables accurate
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.
