Hi Sina,

My guess would be that the substitutions are due to cloning errors although at which step these might have occurred is hard to say. The phusion polymerase is usually quite reliable so i would exclude an error in the pcr.

How do you handle the samples after the PCR for purification of the 180bp fragment? If you run an agarose gel and cut the pieces on a UV table maybe you should reduce the light intensity and the time the DNA is exposed to the UV light. Usually E.coli strains for cloning are deficient in DNA repair enzymes, so any damage by UV light might be carried over to the final plasmid (This might also explain why you have so "many" undirected mutations).

How did the chromatogram from the different sequencing reactions look like? Are they really clean at the sites where you have mutations, or is there maybe a high background so that the program you use interpretes it as a mutation?

Anyways, since you have two positive clones with the wildtype sequence I wouldn't worry and just carry on with these two.

Hi Sascha,

thanks for your help. The chromatogram was very clear in all cases. Sanger sequencing was performed forward and reverse.

I´ve used the PCR product directly (without purification) for ligation (Zero Blunt® TOPO® PCR Cloning Kit) and transformation into JM109 Competent Cells...

I don´t know how frequently PCR or cloning errors occur. Maybe I have to change my PCR or cloning protocol to reduce such errors.

Hi Sina,

Did you sequence these plasmids only ONCE in one direction? I am suspecting that these 'mutants' might not be real mutants but just sequencing 'errors'. When I did my sequencing, for important sequences, I normally sequenced them 2 times from one direction and 2 times from another direction. So, eventually I would get 4 sequencing results. I then aligned these 4 sequences all together, and assembled ('clean') them into one final sequence. So, say, at one position, I might get A,A,A, and A (then A is the correct one), on another position I might get T, T, T and C (then I will know the correct one should be 'T', not 'C'). I have seen the later case occurred in my sequencing results. You may want to try this to double-check to see if those 'mutants' are real mutants.

Hi Yuan-Yeu Yau,

thanks for your answer. I did sequencing in both directions, but only once. So I don´t believe that my results are sequencing errors.

I am not really sure, if my findings are due to cloning/PCR error or true mutations in the analyzed maize kernel.

Please confirm that you have not over-cycled the PCR reactions as over-cycling can lead to accumulation of errors... However, it may also be sequencing error so please check the CHROMAS file of the sanger sequences that you have got..

Good Luck

Hi Sina,

Is this region (180 bp) you are cloning from Maize well studied and documented? It is pretty small.  Do you know this region is 'single copy' in the genome? Repeats?

Hallo!

@Kamal: I´ver already checked the Chromas file. It is okay.

@Yuan-Yeu: Yes, it is well studied and should be a single copy.

I will repeat my experiments and see if I get the same result...

Hallo Johann,

yes this might be an explanation. I have to read about it in detail. Please let me know if you have a literature recommendation for this topic.

Hi Sina, I'm facing a similar problem. Can you tell me what can be the possible reason for point mutation (missense) in the sanger sequencein the process of cloning? It would be a great help

Hi Divya,

after all I think my detected point mutations were real.

I can only summarize some points:

1) If possible not more than 30 PCR cycles

2) Low MgCl concentration, use high fidelity enzymes

3) annealing temperature not too low, touch-down PCR works well

4) PCR product purification before cloning

4) Sanger Sequencing in both directions

Good luck!

Thank you so much for your help :)