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probing mRNA and RT-PCR products

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probing mRNA and RT-PCR products

Postby SarahTessler » Thu Jun 09, 2011 4:22 am

Hi, I am doing revision for my exams by looking at past papers and have this question and no idea how to work it out. Could anyone tell me if my answers are right or a text or website where I could find the answer? Thanks
Questions refering to the image
1) what size mRNA would be detected on northern blots hybridised with PROBE1 - (1kb? exon 1 only)
2) what size mRNA would be detected on northern blots hybridised with PROBE2 - (1kb? exon 2 only)
3) What size RT-PCR product would be amplified from mRNA using the following primers (the primers are labelled as p1-p5, the arrowhead indicated the 3' end):
a)P1+P2 - (none? contains only intron)
b)P2+p3 - (none? the orientation of the primer does not allow synthesis, needs to be ointed towards each other)
c)P3+P4 - (1kb? exon 1)
d)P3+P5 - (2kb? exon 1+exon2)
i would really appreciate some help I'm a distance ed student and the lecturer is not answering my posts or emails...stressing me out!! So thanks a bunch.
Sarah
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Postby JackBean » Thu Jun 09, 2011 6:03 am

both pictures are the same, right?

OK, well, you need to understand, what happens with the RNA after transcription untill mature mRNA is formed. Do you know it?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby SarahTessler » Thu Jun 09, 2011 6:49 am

Yes sorry it came up twice for some reason it should only be the one pic.
umm well it gets its polyA tail at the 3' end and its 5' cap and splicosomes splice out the introns leaving only the exons(the coding regions) which are then joined together. Is that what you mean? I do understand that there will be no introns in the mRNA but another class mate told me that because probe1 does not contain the region with the terminator sequence that transcription will not occur so that the answer to 1) would be nothing. But I am not sure I agree with them. Thanks again
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Postby JackBean » Thu Jun 09, 2011 7:19 am

forget for now about the probes, OK? The cell is not transcripting the gene for some probing ;)
So, you're right, the RNA is capped and tailed, spliced and the exons are joined. So, what will you get in your case?
It will be mRNA, which contains exon 1 (of 1 kbp) and immediately exon 2 (of 1 kbp). But it will be one piece, so if you use probe of any length for any part of the mRNA, you will ALWAYS have 2 kbp band ;)
Similarly, if you use primer4, it won't have any place on mRNA to bind to, thus you cannot have any amplicon.

The rest looks all right ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: probing mRNA and RT-PCR products

Postby SarahTessler » Thu Jun 09, 2011 7:32 am

Oh you are a genius. I get it now. Thank you so much for your time
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