Well, you might have two problems, either you don't have the right PCR conditions (mainly annealing temperature and time) or you destabilized too much your primers...

Troubleshooting needs something to work on... maybe the sequences (wt and mutant)? Your PCR conditions?

I had been using a similar technique for SNPs called tetra-primer ARMS for distinguishing two SNP alleles in a single PCR reaction (using four primers), due to the allele-specific size of the PCR products. Like SAP, to prevent unspecific ligation, there was a mismatch in the SNP-specific primers upstream of the SNP nucleotide, and for this reason the ratio of SNP-specific to common primers was something like 10:1. So, I am wondering whether an increase in the allele-specific primers (forward in your case) can solve the problem. Otherwise, reducing the annealing temperature or changing the MgCl2 concentration may do the trick. 

Thank you for your replies...

Hi Pietro,

I have tied redesigning the primers. Now I've designed a new Rm and Rw to amplify with Fc; which I will check for amplification. I have also tried using gradient pcr for the prev set of Forward primers without any luck. PCR conditions: Initial degeneration (94C for 5 mins), 35 cycles (94C for 45 sec, 53.5C 45 sec, 72C for 45 sec), Extension 72C 10mins, 4C at hold.

Hi Kimon,

Thank you for the suggestion. I will try either by increasing allele discriminating primer conc/ reducing annealing temp/ changing MgCl2 conc.

Hi Gargi, PCR conditions look permissive enough, even without knowing the sequence and the mismatches designed. So if your gradient PCR was not giving any result, I think your choice of redesigning the primers might be good. I see you decided to change strategy, fine, but you could have also tried to change the second-to-last mismatch, which is the most delicate, using the less destabilizing or even just using your point mutation as discriminating, sometimes it works.

Anyway I also agree with Kimon that increasing primer concentration is a good point if your PCR is clean (so you don't get smears or unspecific bands already in the conditions you are using). As a general rule, I regularly use 0.5 to 1 uM conc. of each primer when genotyping on genomic DNA, which is 4-5 times more than for standard PCR. But you could possibly try increasing.

Hi Pietro,

The primers that I designed are in accordance to the SAP PCR protocol which states that if my SNP position is ultimate position, then an intentional mismatch created at the penultimate position with respect to SNP in the the primer helps in allele discrimination. If that is what you were suggesting by second-to-last mismatch, then I guess the trick is not helping me here. I will still try increasing the primer concentration.

Thank you for your suggestions.

Hi Gargi,

that is what I was talking about. Anyway, what I was trying to add to the previous comments is that destabilization of the 3' of a primer is a delicate issue, so I'm not sure which mismatch type you chose for the penultimate nucleotide, but you could try a more permissive one, if the sequence allows it, of course. Or, since it seems that your conditions are very stringent, you could implement the use of the last nucleotide of the primer only to discriminate the alleles, we have one PCR working very well just with this single nucleotide difference between mutant and wt, but you should evaluate the level of destabilization the mutant primer has on the wt sequence and vice versa...

Anyway, I wish you good luck solving this problem!

Thank you Pietro. I suppose this needs several combinations of troubleshooting.

Hi Gargi,

SAP PCR can be performed without the extra 3' destabilization. In most of the case only the 3' end bp itself is enough to distinguish mutant/wild types. 

There are actually two problems in your experiment design: 

1.  PCR product as template. PCR product is not ideal for further PCR process, since more mutations will be introduced in the first round. Can you try genome DNA directly? or maybe a nest PCR?

2. you can try Allele specific primers without the extra mutation at 3' end, which would be a 100% match that will verify whether you have the correct primer or not. The mutation bp is only needed when you have unspecific amplifications. 

Thank you Fang for your response. I understand I will have to try one of these methods to troubleshoot. I used PCR as template only for the standardization because using 3 primers Fc Rc and Fm/Fw failed to give me any product. Hence I was trying with a pair at a time. As suggested previously, I should try redesigning the primers without the extra mismatch in the primer.maybe thats causing more stringency for amplification.

I am also optimizing allele specific primers and getting bands in both alleles even i ran 50 samples simultaneously can any one tell me about trouble shooting