S1 Nuclease Assay | Transcriptome Analysis

[@Jstar001]

Hi, Thank you for this concise and informative video. I have questions with regard to the limitations of this technique. I don't understand what you meant by the last 2 limitations: Why is it not good if regions of perfect U and A mismatch are cleaved, what do you mean by 'regions of perfect U and A mismatch are cleaved'? I also don't get how cleavage is inefficient for DNA opposite a looped-out region of RNA, why would the RNA loop out, and by DNA are you referring to the labelled probe?

[@SpartanTutorials]

Thanks for the kind words Jubair. Ah! I made a mistake in the video, thanks for catching it! It should have been "'regions of perfect U and A match are cleaved" - which should NOT happen. Sorry about that. Regarding the second point, DNA can be looped out if for example RNA is unspliced, and you hybridize it against cDNA. That's one scenario, although I think there can be other plausible scenarios as well. And yes, in this case, DNA will be labelled. The opposite (DNA looping out with RNA) can of course happen in case of genomic DNA-spliced mRNA hybrids.

[@Jstar001]

@@SpartanTutorials I see thank you for your response! I just want to ask, finally, why is it not good for the UA to be cleaved?

[@SpartanTutorials]

@@Jstar001 Sure, that's because A-U is a perfect complementary match between DNA and RNA, and we only want single-stranded, mismtched portions of the two molecules to be cleaved. We want to preserve the region of complementarity to allow us to map the TSS.

[@sudiptajana1854]

nice explanation but still one important concept i feel is missing in this lecture is how the cut out/ restriction fragment of of ds DNA is able is hybridize with its mRNA counterpart.Dont you think we should make ss DNA first using M13 cloning vector followed by hybridisation and all?

[@edwardyhe03]

Yes, have to treat with formaldehyde before cloning into m13 vector

[@wonderfulIndia1]

Plz provides notes of these topic plzzzz