Practice question

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 determination of transcript size, abundance, and promoter-directed initiation efficiency under controlled in vitro conditions.