Hello! I have really strange results in my sequencing chromatograms. The goal was to analyze a point mutation which is heterozygous (DNMT3A gene, in position R882). So I performed a Sanger sequencing with forward and reverse primers (same primers were used in PCR). As a result I got a WT according to Forward primer and a mutation in 1st nucleotide of the aminoacid according to Reverse primer (showed in picture 1).
After that I decided to redo the procedure with the same primers and I got a result, showed in picture 2 (both primers show a double peak, but in 2nd nucleotide of aminoacid!)
So I`m really confused right now, I believe that all that is an artifacts, but I`d like to hear some different opinions and suggestions why does this thing occurs? This doesn`t look like dyeblob or noise, so what else could it be?
Thank you for your answers!
Can you attach the original sequencing files ( .abi files) please and what primer purification do you use for your pcr clean up before running the sequencing reaction please Ekaterina Zaikova
These electropherograms shows two single nucleotide variations (SNV) reported previously, corresponding to c.2644 C>T, p.R882C (1st image) and c.2645 G>A, p.R882H (2nd image) in DNMT3A, and it is incongruent because the two SNVs must appear in both sequences, for this reason, you have to do a third procedure making sure of using the optimal primers and if you want being desalting previously, with the purpose to confirm which or both variants is truly present. @Ekaterina Zaikova
Paul Rutland Here I attach ab. files (C3 and D3 - from the first sequencing, G12 and H12 - from the second).
As for your question after PCR I used Cleanup Mini kit (Evrogen, Russia) and after
sequencing PCR I used Ethanol/EDTA precipitation
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