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Mutation in cloning


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6 replies to this topic

#1 DrLeo

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Posted 25 March 2013 - 06:11 PM

Hi all,
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.
On next day, I chose 2 colonies from the same E.coli population and inoculated separately. After extracting plasmid DNA which contained my target sequence, I sent the samples to company for direct sequencing.
However, there was 1 of 2 sequence results showed a mutation in a unexpected position (new SNPs) although they were the results of the same PCR product.
I am wondering how the mutation could happen, and in which step? How can I avoid getting a mutation like that???
Thank you very much for helping me out this problem!

I attached 2 figure of sequence results.
2 SNPs circled were my target SNPs
The Blue SNP was the unexpected SNPs I got after cloning

Attached Thumbnails

  • ATG5_promoter_hapCA2_chosen.gif
  • ATG5_promoter_hapCA3.gif


#2 bob1

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Posted 26 March 2013 - 12:36 AM

Polymerases have an error rate which can be quite high, but depends on the polymerase used. For example Taq has an errror rate of about 1 base in 10,000 (assay dependent!) due to its lack of exonuclease activiity, but some other polymerases have an error rate as low as 1 base in 1.3 million (e.g. pfu pol). If you used Taq, these "SNPs" probably aren't SNPs but errors during the PCR.

You might be thinking that one base in 10,000 is pretty low, but if you have a 100 bp product, and it undergoes perfect amplification over even as few as 10 cycles (1+2+4+...512=1023 copies) times 100 bp =102300 bases copied... now do you see where the error rate comes in?

#3 phage434

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Posted 26 March 2013 - 05:01 AM

These could also be mis-calls in the sequencing. When you see these errors it is far better to look at the electropherogram rather than the sequence calls.

#4 DrLeo

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Posted 26 March 2013 - 05:49 PM

Thank bob1. But I use the same PCR product for inserting into the TOPO, therefore I think it was not due to PCR process...
Dear Phage434, I also checked the chromatogram but it was totally okay, there was no noisy or other problems.
Maybe in the cutting steps using UV,the mutation occured.
Thank you so much!

#5 bob1

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Posted 27 March 2013 - 01:40 AM

Thank bob1. But I use the same PCR product for inserting into the TOPO, therefore I think it was not due to PCR process...
Dear Phage434, I also checked the chromatogram but it was totally okay, there was no noisy or other problems.
Maybe in the cutting steps using UV,the mutation occured.
Thank you so much!

But that's where you are wrong - each base pair that is amplfied has a 1/10000th chance of being incorrect, which would mean that some of the copies generated during the PCR will have errors, just because it is from the same PCR doesn't mean that there aren't errors in some of the copies!

UV causes CT transversions (in DNA in living organisms i.e. not in extracted DNA!) and double strand breaks - it won't cause your problems.

#6 DrLeo

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Posted 27 March 2013 - 09:49 PM

Thanks bob!
I got your point. Therefore, if I exposed the gel containg the PCR product under UV for a little long time, mutation will not happen?

#7 bob1

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Posted 02 April 2013 - 12:12 PM

Correct, point mutations from UV exposure will not happen in the PCR product, but double strand breaks can, however double strand breaks will not clone or will fail to sequence due to part of it being missing so no primer binding site.




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