What I have heard is the normal Taq Polymerase which we commonly use can amplify a fragment upto 1.5 kb. But yesterday someone told me that the normal Taq Pol can amplify a fragment upto 3kb easily and we do not need to use high fidelity taq polymerase for it? Please suggest.
It will work more or less equally reliably up to 1.5kb. Above 1.5kb the efficiency is going to decline. Some sequences will work, others won't. The workable conditions become narrower, and the yield is going to be poor. Higher than 3kb basically nothing works. So if I had a 3 kb amplicon, I would definitely start out with hi fi. It will make your life much easier.
Just do it and see for yourself. You may be surprised by what you find! One thing I have learned that nothing is more reliable than your own experience. So, if you have time and access to the equipment, this is the way to go.
- It depends to some extent in target secondary structure: if your target has >60-70 % GC residues there will be more propensity for secondary structure and thus your efficiency of PCR will be reduced
- Similarly if your target contains contiguous stretches of GC residues these will stall standard Taq which then detachescand fails to complete synthesis of your full length product; once again reducing PCR efficiency culminating in weaker bands or no large specific bands at all for strong stops associated with runs of GC or AT residues
- This low processivity is an inherent complication of standard Taq and generally means that you obtain less or no product for bands in my experience above 2kb. This is because the larger your target is the more probable Taq will encounter secondary structure or stall at repeat structures and fall away. Moreover adding reagents like DMSO or using higher denaruting temp (>96C) will only reduce these problems partly
- In contrast high processive polymerases like NEB phusion are designed to read through such structures. This means they tend to work more consistently and give you higher levels of product in general for amplicons over 2kb
- Ultimately it depends on what you want to do: if you simply want to see a band to validate a genotype and your target sequence has normal nucleotide content that Taq as indicated may well indeed work. However for higher signal to noise and efficiency in order to isolate the band for say cloning or targets with >60% GC or > 50% AT the single most effective thing you can do is to use a high processive polymerase for such amplicons > 2kb and not Taq
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