I am amplifying 2 kb of a gene. For that i used Taq polymerase enzyme and 22 nucleotide length primer. I checked the amplified sequence by sequencing and it was fine. But when I amplified 2 kb of another gene with 35 nucleotide length primer (infusion cloning), I got many PCR errors (around 5) in each amplified sequence. I tried many DNA samples (10) but still I got errors. I also used exTaq, but still the error exist. I am sure that those were not SNPs. Could anybody share their views about the possible reason of PCR errors?
1. Use high-fidelity DNA polymerase for your PCR amplification.
2. Sequence both directions (F strand and R strand) with 2x each from couple of independent clones to see whether the problem bases are really different or just sequencing errors.
3. Some genes or DNA fragments can be amplified with two 'conserved' primers, but their sequences can be very diverse, such as retrotransposons elements. So, you need to know your gene's characteristics.
That sounds odd. One possibility is that there are more than cone copy of gene or pseudogene in the genome. When you use shorter primers, you get more specific products while the longer primers are more nonspecific, such as being able to amplify another copy of the gene or a pseudogene. Try BLAST the primers and that might help you.
Dear Yunfu Lin,
On both occasion, I am getting specific products. My primers are specific, I already checked them in blast.
Sometimes primers do have wrong sequences which leads to PCR errors.One more thing is the fidelity of taq polymerase is not that good which causes error in pcr products. Therefore, I think you can try pfu dna polymerase which has high fidelity. Good luck
Are your errors identical in each sequencing run? Perhaps they are SNPs and not errors. If you want to be 100% sure, clone the PCR product in a plasmid of your choice and then sequence multiple single, colonies and see if they are identical or not. If you get the same sequence in most cases it is unlikely that your errors were introduced by PCR and more likely that they were present in your template.
1. Use high-fidelity DNA polymerase for your PCR amplification.
2. Sequence both directions (F strand and R strand) with 2x each from couple of independent clones to see whether the problem bases are really different or just sequencing errors.
3. Some genes or DNA fragments can be amplified with two 'conserved' primers, but their sequences can be very diverse, such as retrotransposons elements. So, you need to know your gene's characteristics.
You use pfu polymerase (Invitrogen) to reduce the errors in the PCR amplification.
all the best
Why do you suspect the primers? The errors are in the amplified sequence and not the primer sites right? While you are using different primers than in your first reaction, this concerns a completely different target. How are you sure these are not SNPs?
I think Maximilian Peters made a good point by checking if the errors occur at exactly the same sites in all your samples, or at different sites.
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