3 replies to this topic

[samita]

I just send my gene from mutation PCR for sequencing and have one mising base pair in the sequence....
I have attacged the picture and it seems to me G is there but peak is not visible...
or its deletion mutation from polymerase by itself?????

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[phage434]

I need to see a few more surrounding peaks to be able to tell anything.  And it would be good to know what the before and after mutation sequence was supposed to be.

[samita]

Thanks for your reply...
I have attached the new picture    
alignment
mutant= 5-GAC-TAC-3
WT    = 5-GACCTAC-3
I used the reverse complementary sequence from the sequncing file to align with wild type...

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[phage434]

I can't interpret the upper trace in your picture.  The bottom trace appears to correlate with the called sequence, but the base caller made an error, I believe.  The sequence around the yellow region should read ccgtaggtcct (there is an omitted g, because the g peak does not appear to be quite wide enough, although from the spacing it is quite clear that there is a second g.

I assume this is the problem you are worrying about -- although I still can't make any sense out of the top trace.
If I read things correctly, the answer is "don't worry, be happy."