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Point mutation in sanger sequencing


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#1 Preziosa Buonocore

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Posted 10 November 2015 - 09:05 AM

Hi guys.

I performed a sanger sequencing on both a cDNA and a DNA from the same sample. I found a point mutation in the cDNA but not in the DNA. 

 

Have you ever happened something like that?!



#2 labtastic

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Posted 10 November 2015 - 10:01 AM

The first thing to check is the sequencing itself.

 

It is not unusual for the computer to call the wrong nucleotide. Check the raw trace to see if the "mutation" in the cDNA is real before doing any other kind of troubleshooting.


Edited by labtastic, 10 November 2015 - 10:01 AM.


#3 phage434

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Posted 10 November 2015 - 01:38 PM

Post-transcription mutation of mRNA is rare but not unknown. But I agree that first step should be looking at the trace of the sanger sequencing, and likely repeating it. PCR errors either in the initial RT-PCR or in subsequent amplification could explain this.






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