The RNA component is typically short 159 bases long in Tetrahymena, and 451 bases long in humans, though 1.3 kb in yeast

TLS Online TPP Program

#Id: 2636

#Unit 13. Methods in Biology

S1 mapping the 5’-end of a transcript

TLS Online TPP Program

#Id: 2637

#Unit 13. Methods in Biology

Primer extension Using primer extension one can locate the 59-end of a transcript by hybridizing an oligonucleotide primer to the RNA of interest, extending the primer with reverse transcriptase to the 5’-end of the transcript, and electrophoresing the reverse transcript to determine its size

TLS Online TPP Program

#Id: 2638

#Unit 13. Methods in Biology

One simply harvests cellular RNA containing the transcript whose 59-end is to be mapped and whose sequence is known. 

TLS Online TPP Program

#Id: 2639

#Unit 13. Methods in Biology

Sometimes the S1 nuclease will not digest the single stranded regions completely, so the transcript appears to be a little longer than it really is, But another method, called primer extension, can tell the 5’-end (but not the 3’-end) to the exact nucleotide

TLS Online TPP Program

#Id: 2640

#Unit 13. Methods in Biology

Run-off transcription is a means of checking the efficiency and accuracy of in vitro transcription.

TLS Online TPP Program

#Id: 2641

#Unit 13. Methods in Biology

The size of this run-off transcript locates the transcription start site, and the amount of this transcript reflects the efficiency of transcription.