I am getting the desired amplification with normal DNA Taq polymerase but I am not getting it with high-fidelity Taq polymerase, I am getting non-specific amplification. so can I go for sequencing with the PCR product which is amplified by normal taq polymerase? and what may be the error rate when compared to the high fidelity Taq Polymerase?
People have been sequencing normal taq pcr product for many years.Taq Errors are quite random so occur all along the amplimer so only give a very small sequence signal at all positions compared with the correct sequence. Hi Fidelity enzymes are more useful in cloning where there is a need for exactly the correct sequence. The error rate for taq is about 1 base in every 13000 but modern hi fidelity mixes will only make 1 error in about 660000 bases. An efficient pcr could produce 1000000 copies in 20 cycles so there will be errors present in your amplifications but not many
a Good Sanger seq sequence has a phred score of 20 (1% error rate). So the readout is much less precises than taq Polymerase. i did honestly never saw a mismatch due to PCR
Thank you so much for your valuable suggestions Paul Rutland , Sven Reister
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