Section Bank Bio #31

[wheatthinners]

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[yeasureyoubetcha]

On the correct answer (A), I'm confused as to why you would need 2 primers to create cDNA. Wouldn't you be able to just put a primer on 4 and have it synthesize the cDNA transcript from the mRNA all the way through? Also, doesn't it only synthesize from a 5' to 3' direction, so putting a primer on the 1 (since mRNA will be flipped) would be essentially useless since it will transcribe not much at all. What am I missing here?

Thank you!

You need a forward and a reverse primer to amplify what is between said primers so you have both the template and non-template strand (DNA is complementary) to continue successive rounds of PCR.

Take mRNA extract and RT-PCR it to get the cDNA. As the explanation says, "due to transcript length of both isoforms, it must contain exon 1 and 4 in every transcript." By using say an E1Forward primer and an E4Reverse primer, you will amplify all isoforms regardless of splice variant which you can then visualize on the gel.

 

[wheatthinners]

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[Thecomposer]

The only reason I have an issue with answer A is that you will NOT notice a difference between only 3 base pairs when running a gel. Because the isoforms are so close, you need to probe the specific exon, which is why I liked B better. Choice A only works whenever you have a nice enough distance between base bairs. I mean...if you had a super highly concentrated gel and had like 30 hours to run it, then maybe you can see the difference if you had a cadillac-quality DNA marker....oh well.