Somewhat of a guess but a low fidelity polymerase could incorporate mismatched bases in your initial amplicon that prevented nested primer binding in the second PCR run.

How are you detecting the variation - is there a size difference (eg detected on a gel) or are you detecting differences by HRM? Either way, do you mean the variant fails to amplify with Platinum Taq or you cannot differentiate the variant from wildtype when using Platinum

Definitely some more information is needed to be able to answer... On the lines of Paul's answer, are you talking about an SNP or a structural variation (deletion/insertion, inversion), and are you working on a cDNA or on genomic DNA, and what is the reason for a nested PCR, enhancing sensitivity or specificity of your amplification? Is the variant in the primers or does it come in the middle of the amplification? are you then sequencing or how do you detect the variant?

In any case, it is recognised that different polymerases do behave differently, some have preference for certain amplicons which others don't, some are more efficient and some less, some produce more unspecific and some less... Sometimes problems are solved by adapting the protocol to the specific Taq... so the fact that you don't change the amplification protocol is not necessarily a positive thing in this case. A different polymerase may require a different annealing temp or time and/or a different elongation time and/or a different primer concentration and/or a different Mg2+ concentration to perform as well as another...

Sorry, I should have provided more info.

We are working on PKD1 gene which has 6 pseudo genes in Human Genome.

We were provided with some Positives from another lab for Validation.

One positive of the one Long Range fragment is picked up when we using   KAPA MM (with Robast HotStart DNA Polymerase) for nested PCR and not picked up by PCR MM containing Platinum Tag. It is a duplication of 18 nt.  I believe that if Mutation is true any TAG should pick it up.

What do you think about it? Thanks

Hello Anna,

as I said previously, not necessarily... Here is why: the PKD1 pseudogenes are located in a different chromosomal region (16p13.1) than the proper gene (16p13.3), with possible different chromatin or other regulatory arrangement. Depending on how you extract and purify the genomic DNA, the region might be more or less accessible in certain buffer conditions, which are set by your different master mixes. That's why with different polymerases and mixes you might get different results, indeed... it's not a surprise!

The primer design strategy might also favour this situation: it is possible that with a different primer design you might overcome the problem. As well as with a different gDNA extraction method: as a general rule, the cleaner the gDNA, the less variability in PCR results, which means a good digestion of proteins with Prot. K and an old style extraction with Phenol/Chloroform at neutral pH followed by flocculation/precipitation in ethanol is always a good practice... except that is a bit dirtier lab practice, might take more time than the quick kits and might need more starting material... The reason is that nucleic acids in cells are never alone, they are covered by interacting proteins (of course histones, but also many others from the transcription machinery, or the repression regulatory mechanisms, or repair systems, etc that protect them from accidental degradation by nucleases) and these proteins not properly removed then affect, even in a reaction mix, DNA secondary structure, primer annealing and/or Taq polymerase elongation efficiency and processivity. Depending how the mix is composed (that's one of the differences between KAPA and Platinum kits, besides the Taq polymerases themselves), you might have better or worse results in certain PCR reactions... Apparently you are in one of those situations...

Remember, PCR is powerful, but VERY tricky, and you need to have clear in your mind what's behind it. Have a look at the composition of the master mixes, see what changes (salt type, salt concentration, Mg2+ concentration, pH, additives (typically glycerol)...) and search how each parameter affects the many events happening in a PCR reaction (DNA denaturing efficiency and time, primer annealing kinetics and thermodynamics, Taq activity, Taq processivity...) and you might be able to figure out why using two different mixes and polymerases you have different results...

Good luck.