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Testing primer specificity...


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#1 missalga

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Posted 05 October 2009 - 07:58 AM

Hi,

I wondered if anyone could help me out with their thoughts on primer design:

I have a nice alignment of my group and its outgroup, and have made supposedly group-specific primers (so blast tells me). But I am going to be using them on mixed environmental DNA, so I need to make sure that, practically, they aren't going to pick up anything/everything else. Unfortunately, I don't have any gDNA from my target group or outgroups, so I can't test them this way (by raising the PCR temperature until I don't get a band in the outgroup, while still getting a band for the target group).

My only idea so far is to try the primers out by doing PCRs on environmental extractions (which I assume contain my target group) at increasing temperatures, and taking the highest temperature that I still get a band at, and then clone the gelcut and colony pick. Hopefully, I'll only get target sequences.

Does anyone think that's sensible? Is there a better way?

Thanks very much for any help,
M.A.

#2 ShannonJ

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Posted 05 October 2009 - 09:20 AM

That's probably the only way, followed of course by sequencing those PCR products. You could do a negative screen, using a primer-free master mix and testing non-target organisms to at least ensure that you won't pick up common contaminants or other non-target organisms. Sorry.


Suggested PCR reactions:
Organism 1 Reaction 1 - general primers, non-target organism
Organism 1 Reaction 2 - specific primer, non-target organism

You might try with a few different non-target organisms to increase your confidence...
Good luck!

Hi,

I wondered if anyone could help me out with their thoughts on primer design:

I have a nice alignment of my group and its outgroup, and have made supposedly group-specific primers (so blast tells me). But I am going to be using them on mixed environmental DNA, so I need to make sure that, practically, they aren't going to pick up anything/everything else. Unfortunately, I don't have any gDNA from my target group or outgroups, so I can't test them this way (by raising the PCR temperature until I don't get a band in the outgroup, while still getting a band for the target group).

My only idea so far is to try the primers out by doing PCRs on environmental extractions (which I assume contain my target group) at increasing temperatures, and taking the highest temperature that I still get a band at, and then clone the gelcut and colony pick. Hopefully, I'll only get target sequences.

Does anyone think that's sensible? Is there a better way?

Thanks very much for any help,
M.A.



#3 swanny

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Posted 05 October 2009 - 05:20 PM

Touchdown PCR should just give your organism, because only 100% homology targets will amplify first, and out compete other sequences that prime at lower percentage homology.

Also, gradient PCR will show you different organisms that generate products and the annealing temps that do so. Presumably some of the products will be from your organism and others from non-target bugs.
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#4 missalga

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Posted 05 October 2009 - 06:32 PM

Great, thank you very much for your reply Shannon.
It's good to know I'm not missing something obvious that's maybe done in other fields. Thanks.

Edited by missalga, 05 October 2009 - 06:35 PM.


#5 missalga

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Posted 05 October 2009 - 06:34 PM

Touchdown PCR! That sounds brilliant. I'd never heard of it, but I think it might definitely be worth trying. Thank you for alerting me to it.
:(

#6 gebirgsziege

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Posted 05 October 2009 - 10:40 PM

if your primers are specific, you should be able to do direct seq from your environmential sample.

Do you have any clones containing the region of interest of your organism? then you can spike samples with your target region (i.e. clones).....
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#7 missalga

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Posted 06 October 2009 - 11:41 AM

I can't do direct sequencing because it's a target group, not single organism, so I'm expecting hundreds of taxa within the group in some environmental extractions... and no, I don't have any DNA from any of the group members!

It's a toughy.




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