I think Paul has the right idea. A particular lab may have used a given annealing temperature, but if you are not using the same exact same kind/brand polymerase and all the same buffers, salt concentrations, etc... then the annealing temp might be different for you. When working with new-to-you PCR primers, the ideal annealing temperature must always be experimentally determined by a given user. The correct way to do this is a temperature gradient. (For a primer you thing will work at 52C, I would try a range of 50-57C.) Use the highest annealing temperature from the gradient PCR that gives good amplification for a set of primers . If your two primer sets (allele 1 and allele 2) have very different annealing temperatures, you many need two different PCR reactions, one for each primer set.

Two other things I would recommend: 1) I would reduce how much primer you are using. Maybe try using 1/5-1/10 as much. Excess primer can cause non-specific priming. Check the directions that come with your polymerase for ideal amounts for that enzyme. 2) Unless you are starting with very low concentration template, I would skip the "common PCR" you are doing first. It could be part of your problem.

Last ditch effort, cut out bands or purify PCR products and send them for sequencing to see what they are. A band at about the right size does not guarantee is it the right product. Sequencing will always tell you exactly what you amplified. Best of luck!

there may be a temperature difference between the 2 thermal cyclers or the salt concentrations of the pcr mixes making direct comparisons difficult so if possible run a temperature gradient of the 2 allele specific second amplifications as it looks like your annealing temperature is so low that both primers can anneal to all sequences so everything will look heterozygous.Ideally run the gradient on a homozygous normal and a homozygous mutant to make interpretation of the gradient easier.

Then address the problem of your primer dimer which may indicate that you are using too much primer

I think Paul has the right idea. A particular lab may have used a given annealing temperature, but if you are not using the same exact same kind/brand polymerase and all the same buffers, salt concentrations, etc... then the annealing temp might be different for you. When working with new-to-you PCR primers, the ideal annealing temperature must always be experimentally determined by a given user. The correct way to do this is a temperature gradient. (For a primer you thing will work at 52C, I would try a range of 50-57C.) Use the highest annealing temperature from the gradient PCR that gives good amplification for a set of primers . If your two primer sets (allele 1 and allele 2) have very different annealing temperatures, you many need two different PCR reactions, one for each primer set.

Two other things I would recommend: 1) I would reduce how much primer you are using. Maybe try using 1/5-1/10 as much. Excess primer can cause non-specific priming. Check the directions that come with your polymerase for ideal amounts for that enzyme. 2) Unless you are starting with very low concentration template, I would skip the "common PCR" you are doing first. It could be part of your problem.

Last ditch effort, cut out bands or purify PCR products and send them for sequencing to see what they are. A band at about the right size does not guarantee is it the right product. Sequencing will always tell you exactly what you amplified. Best of luck!

Paul and Heather,

Thank you for your help. I really think it is the low annealing. Promega has a site that lets you put in the primer and tells you the Tm for their particular enzyme mix, so I am going to do that with these and then try an annealing 5 degrees lower. Only because I need results for this particular part asap. BUT if it still doesn't come out I will try to run a gradient. The two forward primers differ only at the 3' base which is the SNP difference.

Taq is not supposed to be able to extend if the 3' base is not completely hybridized with the template so I'm really thinking its a temperature issue...I hope...

Im using a 10uM primer stock and using 1 ul in 25ul reaction so its like 0.4uM or something of that nature. I didn't see primer dimers on the gel...wouldn't they be small bp size and come up under my amplicon bands?

Thanks again. Ill update tomorrow

I think it will end up quicker to run the gradient. TM calculations tend to be conservative in that they give a temperature that will always work but in this technique you may have to choose a sub optimal temperature for one primer to ensure that the other primer does not work at all. Do not forget also if using the online site that one base will not bind the template. I was wrong about the primer dimer it does look more like non specific amplification and may get better with increased annealing temperature. Taq will extend from mismatched primers if the temperature is low enough....the mismatched end waves around and even if the end momentarily touches the mismatched base on the template the extension speed of taq is approx. 120 bases a second and so extension will occur then you have perfect amplimer so amplification will continue.

good luck

Paul

Paul,

Is that possible to do if I dont have a positive control that I know for sure is homozygous for either? And do you also agree with Heather that I should skip the first amplification? The first amp is a ~600 bp region that contains the sequences of the allele specific primer which when amped is 216bp. She suggested I just do the allele specific from the genomic dna directly

also, Heather said "unless I have very low starting concentrations of genomic"....my samples range from 3-26 ng/ul so they are relatively low. However, I did a serial dilution and found that I could detect all of my genes of interest, including this one, at 0.625 ng in a reaction if I did 40 cycles.

Hi Jory,

I think you should be ok with a known heterozygote and a normal sample. Running a gradient on these 2 samples with both primer sets should give you temperatures at which both primers can be used with the other primer not amplifying hopefully.

Heather's advice is good as expected from someone who runs a quality laboratory and I think that she is rightly concerned that amplifying across the polymorphism and then re amplifying creates huge amounts of amplified product increasing the probability of pcr contamination . My feeling also is that unless the allele primers work very badly on genomic dna or you have only tiny amounts of sample dna then the first amplification is not necessary. I would prefer to see amplification at less than 40 cycles which is a huge amplification and indicates that improvements can be made somewhere in the process ( dna prep perhaps)as that is approximately 10exp12 times more amplified product

Ok I am taking both advices and my own idea and combining all for today. Raised temp, lowered cycles to 35, ran genomic dna and pcr product to see the differences. If this doesn't work I will run gradients tomorrow on genomic dna if it the genomic runs well today. My problem with trying the gradient today is that none of the samples are known to be either homozygous or heterozygous for sure.

thank you both for reply. Ill update with a new gel pic tonight

ps. only trying the pcr product again because i had just enough left to do it. I will just use genomic from now on if you both agree that is the best idea. I really only did it this way because it is described to do it this way any time you look up allele specific pcr unless you use that other method where you change the length of the specific primers by 10 bases to see the separation but I hadn't read about that method until I'd already ordered the primers.

I also used a gel purified pcr product not just a direct one if that makes a difference to the opinion on using the pcr product

Hi Jory, Paul is correct. Even the best primer Tm calculations are estimates. With only one base different, even if it is the 3' base, the Tm used needs to be pretty stringent in order to have each primer only extend on the correct template. Some other methods that might improve specificity (other than using a higher annealing temp) are: 1) reduce the amount of primers in the master mix, 2) reduce the amount of dNTPs in the master mix, or 3) add 1uL of DMSO to the PCR reaction (which will alter the annealing temp, so that may need to be re-tested). Good luck!