Aren't they supposed to be the same height for the same nucleotide base?
Hi sarah
sanger sequencing data depends on quality of the amplification. as for individual peak the height depends on, first the nucleotide (each associated dye has different emission strength, and second the number of the fragments containing and ending by this base will be amplified and sequenced.
now, in your case since you got a variant, you will also have to consider the overlapping of the 2 dye emission lengths AND the proportion of C and A in the sequence of your sample (frequency of the variant).
I hope this helps
fred
Hi sarah
sanger sequencing data depends on quality of the amplification. as for individual peak the height depends on, first the nucleotide (each associated dye has different emission strength, and second the number of the fragments containing and ending by this base will be amplified and sequenced.
now, in your case since you got a variant, you will also have to consider the overlapping of the 2 dye emission lengths AND the proportion of C and A in the sequence of your sample (frequency of the variant).
I hope this helps
fred
Frederic's answer is very good. I've attached a brief guide from our core facility that links sequencing problems with associated electropherograms. It has helped me troubleshoot problems with my own Sanger sequencing projects.
in the early days of Sanger sequencing enzymes were not as good as now and magnesium was used instead of manganese when extending and adding the dideoxynucleotide. Then the differences were very clear, For instance a G after another G was always stronger so a run of Gs just became stronger. There were also recognised 2 base sequences where the next base was either very weak or stronger than the base before so addition of bases was sequence dependent, I think that even with better enzymes there is still a slight remnant of this sequence dependent addition remaining
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