RNA polymerase can initiate a new RNA chain de novo on a DNA template and thus does not need a primer

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#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

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#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. 

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#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

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#Id: 2640

#Unit 13. Methods in Biology

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

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#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. 

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#Id: 2642

#Unit 13. Methods in Biology

Notice that S1 mapping and primer extension are well suited to mapping transcripts made in vivo; by contrast, run-off transcription relies on transcription in vitro.