Looking for a gene to start with when doing PCR

[Sina Amoor Pour]

Hello,

I am one of the organizer of Stockholm Makerspace. During the spring we moved to a new location and got the opportunity to build a wet lab. Through a network of people and a lot donations from life science companies, universities and cooperation with the local iGEM team we have managed to build a lab with all the equipment needed for at least PCR, transformation of bacteria and simple light microscopy. Now we are in talks with a couple of companies selling reagents and primers. All that is needed now for us to do our first PCR experiments is a gene that we can amplify. I'm looking for a gene that would be fun for people that have never been in a lab to amplify and look at with gel electrophoresis.

I have been thinking about doing the cilantro test. But I'm not sure which gene to test for. Is it the OR6A2 gene that should be amplified? And if you have it you should hate cilantro and if you don't have it you should love cilantro (bascially)? If not, which gene is the cilantro-hating gene?

And do you have any other suggestion for other fun genes that we can try out?

All suggestions and advices are appreciated!

Thanks,

Sina

[Kermit Henson]

Hi, 

ACTN3, for example

take a look

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043132#s2

With a PCR you just go for an insdel site. easy to see with sybr

example = C/T CAT->TAG QLN->STOP

Also you can look for STR sites

If you are looking for the Rs72921001 (http://snpedia.com/index.php/Rs72921001), you'll need to use a qPCR or Sanger. With a "normal/regular" electrophoresis you cant distinguish nucleotides. 

[Sina Amoor Pour]

Thanks for good information. So the only things that can be checked with PCR are indels and STRs?

Could you also elaborate on this "example = C/T CAT->TAG QLN->STOP". I didn't really understood what that meant.

[Kermit Henson]

With a PCR plus an agarose gel you can only check the length of a DNA fragment, you CANT check differences in 1 nucleotide (for example the change of an A for a C). So, normal PCR is useless for checking SNPs (https://en.wikipedia.org/wiki/Single-nucleotide_polymorphism).

The example example "C/T CAT->TAG QLN->STOP" is to show how works the mutations you should look for to check with a PCR. That's, the change of a dNTP change your "gene", so you get a STOP codon instead of glutamine. Sorry, I realize that this example may not be the best :/

With PCR you can check the existence of a DNA fragment in your sample. So, you'll need primers that sorrund the target zone. If you replicate that zone, you'll always get the same lenght fragment and you wont be able to check it by electrophoresis. 

My proposal is that you look for genes that can get insertions or deletions, like the ACTN3. So, when you try to replicate the ACTN3, you'll get a fragment if the sample contains the INS allele. After this, you can check by electrophoresis if the sample contains a band of "XXX bp", which will be the size of the INS allele.

For STR (https://en.wikipedia.org/wiki/Microsatellite) regions it works quite similar.

In the paper I linked, it explains the primers and PCR conditions. You can order the primers or buy t directly to biorad or lifetech.

--

Kermit Henson

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[Sina Amoor Pour]

Thanks, make sense. I will take a closer look at that paper and see if we can test the ACTN3 gene.

[Josiah Zayner]

I hate to sound like a bot but you should check out The ODIN http://the-odin.com

We have a kit for bitter taste receptor genotyping that includes a protocol, primers and taste test strips. Though I don't know how well the enzymes will survive during a trip to Sweden, maybe I can give you a discount to test it out?

Thanks,
     Josiah Zayner

[Kermit Henson]

This kit http://www.the-odin.com/human-dna-cheek-swab-purification-kit-and-genotyping-primers/ seems nice to me. 12 samples @ 20 usd, that makes 1.7usd/sample, you just need to add taq and mastermix.

In this case, he is using an enzyme to cut the pcr product, so you can see 1 or 2 bands depending on the allele. Another solution for the same problem :)

[Sina Amoor Pour]

Josiah, I looked around on the-odin.com. That test for bitter taste receptors seems interesting. I tried to make a checkout to get an estimate on the shipping cost but it said that you don't ship to my address. If you can get me a nice price on the shipping cost I will get one and try it out. I say might because there are customs prices that will be added too and I have to check how much that is. But lets start with the shipping costs. How much will that be?

[Josiah Zayner]

Sure. Can you send an address to j...@the-odin.com

Thanks!

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