What may be the possible reasons for getting non-specific DNA amplification which is amplified by high-fidelity taq polymerase? Even though we get good desired amplification by normal Taq polymerase.
Non specific amplification usually happens when the primers lack specificity and anneal to many places. This can happen when there are divalent ions like Mg present and the annealing temperature of the primers is lowered. It may be that the pcr buffers of the two enzymes are different and that the magnesium in the high fidelity buffer allows the primer to anneal non specifically and produce other amplimers. Hi Fi enzymes tend to give a highly accurate amplification but not necessarily high specificity because the specificity is determined by the primers not the enzyme
Hello Subrahmanya Hegde ,
do you use the same parameter when doing your PCR with the normal taq vs. the high-fidelity ?
In addition to what Paul Rutland said, for the same primer pair, you don't necessary calculate hybridation temperature the same way depending on the polymerase you use !
I would suggest to run your PCR again while increasing Tm temperature by 5°C. It will tell you if it is a prime specificity problem.
Moreover, having multiple bands is never great but if you know the size of the amplicon you expect to obtain, you can totally purify your band of choivce from a well separeted agarose gel !
P.S.: For your other question, I assume you talk about SANGER sequencing of PCR product, yes you can totally sequence PCR product amplified with normal Taq ! The standard procedure is to sequence your product in both direction with a forward and a reverse primer (you can use the same one you used for YOUR PCR).
Good luck,
Philippe.
Hello Philippe Paget-Bailly ,
I altered the annealing time (5sec) instead of normal 30 sec Regarding the use of PCR parameters for high fidelity, but I kept 30 sec while doing PCR with taq pol and I got desired amplification, and I used the same for High fidelity as well, but I got non specific amplification, then I went through specifications of high fidelity, It was mentioned that to avoid non specific amplification set the annealing time for 5-15 sec. I did the same but I didn't get my desired band at desired range.
BTW thank you so much for your precious valuable suggestions!!
Regards,
Subrahmanya Hegde
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue