hi to every one,

i am trying to amplfy a region in NKX2.5 gene for mutational studies using ASO PCR technique, but the problem is pimers i have are not of same size. one is 17 mers another is 24 mers. if anyone can help me out to optimise the annealing temp for this pcr would be helpful.

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Hi!

Well, what did you try so far and what are the actual annealing temperatures calculated for your primers. I don´t see a problem in your 24-mer but 17-mer is a bit short for genomic stuff, as it might be somewhat unspecific, especially if you have to keep temperatures low.

Without knowing your protocols I would suggest two things:

1) gradient PCR with the calculated temperatures for your primers with a range of +2 °C- -5 °C
2) if you have some information from gradient PCR you can do a touch-down PCR.

Hope this input is a start.
Cheers

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Can't you make your 17-mer primer a bit longer? Just add some extra bases (preferably of course matching your target sequence).

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I agree, 17 is a little too short for genomic DNA... what are the Tm's of yr primers??

usually a good starting point is 5degrees lower than the average Tm of the primers. or s gradient like Bomber suggested

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these primers iam using for Allele specific oligonucleotide PCR, where u have 2 primers to detect one mutation viz.. Wild type primer and Mutant primer both of these primers are forward primers, they have on e common reverse primer. if there is a mutation in the genomic DNA the mutant primer will anneal if there is no mutation in there the wild type primer will anneal.

for this the mutation has to be either

1. at 3' end so that it will not anneal or

2. it has to be at 5' end so that it will not extended by enzyme.

but the article iam following has listed the primers with mutation in between the primer, i couldnot understand what is the logic for having mutation at middle of the primer.

i hope it is clearly understandable

help will be appriciated

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Well,

maybe I´m totally off the track but here is what I would do:
Design primers 5´ and 3´ of your region of interest do a pcr and sequence. you will get heterogenous sequence if you have a mutation on either allele. If not it is one clear sequence.

I agree that a mutation in between the primer is somewhat strange, as you will probably get annealing especially if you have strong 3' binding, thus making the ASO PCR somewhat unreliable. It reminds more to side-specific mutagenesis having a mutation in the middle of the primer.

Anyway the suggestions given before are things to start with..., especially a gradient PCR should give hints about optimal PCR conditions in your case.

Good luck
Cheers

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thanks mate for the consideration

actually iam trying to screen 500 samples for the known mutations in NKX2.5 gene, and i cant really have all of the samples sequenced for the study i think its a bit expensive way of doing the things, that is why i have chosen ASO PCR for some of my mutations, and RFLP for rest of them. but i would go for sequencing to screen unknown mutations in this perticular genotype to see the variation in the prevalence of mutations compared to europe.

so do u want to say that inducing a mutation in 5' or 3' end of ASO primer could yeild better results than mutation in middle of the primer.

i could not get u about side specific mutatgenesis related to having a mutation in middle of the primer.

thanks again..

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