When I did cloning, after getting the PCR product of the target sequence, I inserted it into "TOPO I" vector and then transformed into competent cells. The next day, I chose two colonies from the same E. coli population and inoculated separately. After extracting plasmid DNA which contained my target sequence, I sent the samples to a company for direct sequencing. However, there was one of the two sequence results that showed a mutation in an unexpected position (new SNPs) although they were the results of the same PCR product. I am wondering how the mutation could have happened and in which step? How can I avoid such mutations?
It is possible that the sequencer misread that nucleotide, Check the chromatogram manually to eliminate this possibility. Also read the sequence using both forward and reverse primer, compare the result to check whether the mutation that you are seeing in forward (primer) read also appears in the reverse (primer) read? this would give you the answer whether mutation (point mutation) is due to ambiguous sequence read or due to an error by the polymerase during PCR amplification. In the latter case choose the clone which doesn't harbor any mutation.
Thank you very much, Moorthy!
But I checked the chromatogram for sure and there was no noisy or any other problem at that position. I also used 2 primers to read the sequence. I think it is not due to the sequencing...
Also, I used the same PCR product for cloning, therefore, I think the mutation happened in the steps after PCR.
In other steps of cloning except PCR and sequencing, can the mutation also happen?
Thank you again!
PCR can introduce mutations. That is why you need high fidelity polymerase and limit the cycle numbers for PCR.
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