An NPC is made up of many copies of some 30 different proteins called nucleoporins.

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#Unit 13. Methods in Biology

The probe must span the sequence where the transcript starts or ends. After hybridizing the probe to the transcript, one applies S1 nuclease, which degrades only single-stranded DNA and RNA.

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#Unit 13. Methods in Biology

Double-stranded DNAs, RNAs, and hybrids are protected from S1 nuclease degradation. 

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#Unit 13. Methods in Biology

S1 mapping the 5’-end of a transcript

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#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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#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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#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