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how to get specific amplfication of a gene that is extremely similar to another - (Jan/21/2007 )

I have to amplifiy a gene that is extremely similar to another gene using PCR and there is only one mismatch in the region that I can put my primers. I have tried putting this mismatch base pair at the 3' end of the primer, but I'm still getting amplification of the other gene that I don't want. angry.gif

What can I do??? unsure.gif

-Floz-

Well, you've done what I would have suggested. You could try a gradient PCR to see if there is a temperature where the mismatched primer fails to amplify. Are you sure the other gene is there? Another approach would be to find a primer outside of the coding region and amplify using that. You might be able to find that sequence with inverse PCR, if you don't already know it.

-phage434-

what about the reverse primer in the set? if it is specific then you can select for your gene with it.

-mdfenko-

Thanks for the reply.
Sorry this is probably a stupid question, but what is inverse PCR?

QUOTE (phage434 @ Jan 23 2007, 03:24 AM)
Well, you've done what I would have suggested. You could try a gradient PCR to see if there is a temperature where the mismatched primer fails to amplify. Are you sure the other gene is there? Another approach would be to find a primer outside of the coding region and amplify using that. You might be able to find that sequence with inverse PCR, if you don't already know it.

-Floz-

Inverse PCR is used to locate flanking regions of a known sequence. The idea is to cut the genomic DNA with a restriction enzyme, and then circularize the resulting fragments. PCR primers are made which are directed outward (away from each other) on the known sequence, and are used to PCR across the circularized fragment, providing flanking sequence. Since you have two different genes, with luck your enzyme will cut with two different length flanking sequences, and the two PCR products can be separated on a gel and then sequenced. A variety of enzymes can be chosen for this technique (check that they don't cut the sequence you already know). For large genomes, nested PCR can be effective during the inverse PCR process.

-phage434-

The nested PCR option is a good idea. i.e. design primers that anneal to unique sequences outside your sequence of interest (but not the other sequence) and amplify your sequence from that template once you've isolated it.

-killerkoz17-

on the topic of inverse PCR,

i am told that the vectorette PCR system gives better results then inverse PCR. does anybody have any opinion on this matter? or better yet has used the vectorette idea.

-perneseblue-

Maybe this is too simple minded, but have you looked for a restriction enzyme cutting specifically only the non desired product? You could cut the one you don't want and separate the fragments by electrophoresis.

K.

-kr├╝melmonster-