Thread: genomics question

Describe an experimental strategy to determine whether an mRNA from mouse gene X is specifically type I (and not any of the other types). Give your rationale and show hypothetical results in your answer.

I cant think of a way to figure it out. I was first thinking of doing a an RT-PCR experiment and using polyacrymalide electrophoresis to see the difference in lengths, but im not too sure as to how good that answer would be. My second thought was to do a northern, but that would be just as effective as PCR.

Would it make sense to use each of the 4 mRNA's as probes and using a high stringency setting and see if they hybridize? i suppose that would be using a northern blot method.

I was also thinking of using some sort of way to express the proteins that are made from this "Gene X" and then check the functionality of it, but i dont know of any procedure that does that. Is there one?

Am i on the right track? how else would i answer this question. I am not too familiar with micro array, so if anyone knows what that is and can explain to me how it works that would be greatly appreciated.

Do two PCRs with primers designed to recognize 1A and 3.

A northern blot for 1A and 3 would work also, but would be more time consuming than PCR.

Wow thanks for such quick replies! So when i have a primer for 1A and 3 then i'd have only one PCR product that would contain 1A 2 and 3 right (so it would be shorter than the whole mRNA product but it would confirm the gene)? So i'd take the mRNA of the mouse and use 1A and 3 as the forward and reverse primers and if a PCR product is formed then its conformed that its its type 1 and if no PCR product is formed then we know that its not type 1, correct?

<This post was a mistake>

no the picture is the mRNA, but i dont understand how you would be able to predict the lengths by doing the 2 seperate PCRs?

Because I'm assuming the numbers represent separate exons, so you know that the length is likely the same as that in the mRNA.

Edit: I'm now thinking it's better just to go with a Northern and that way you can just talk about the mRNA. Or, isolate the mRNA and PCR that .

but if you were to do a PCR for 1A how would you know where the PCR product would end and same for 3 if we designed the primers to be 1A and 3? could be perhaps do a micro array in a high stringency setting and use 1A and 3 as oligomers and if they both are present it means that dna contains that gene?

The picture gives you the length of the mRNA, (200 bp for an inch in the picture I think), you PCR the mRNA from 1A to 3, you expect around 600 bp length. You run it with a ladder of known length to give you an idea of how long the segment you did is.

Edit:
1) If it is not type 1 you don't expect to get a product at all.
2) If you get a product from the PCR you expect it to be around 600 bp, which you confirm with a ladder.

OH okay, that makes sense! thanks a lot! you've been very helpful!

Originally Posted by humaders99

OH okay, that makes sense! thanks a lot! you've been very helpful!

It took me long enough to figure it out, sometimes it's worth talking it out haha.

I had a brain fart because I thought we were talking about DNA, and I didn't register the RT-PCR in the first post .

You can design the primers to get any length of DNA you want.

You could also have a reverse primer at the end of 4, and two different forward primers; one in the beginning of 1A and one in the beginning of 1B. Then the size would immediately be a give away.

But usually you try to get primers with optimal characteristics for a standard PCR reaction so I would still do two PCR reactions. One to determine if 1A is in the sample, the other if 3 is in it.

Combining the data should tell you what it is.

Or just send your DNA away for sequencing!

can you explain what primers i would use if i did them separately and what results i would see if i ran it on a gel?

can't tell you without knowing the sequence.