sequencing problem-one double pick

[Anonymous]

Hi there,
I was wondering if anyone could help me to understand this problem: I am sequencing a PCR purified product containing LoxP sites. The Lox P is 34 bp consisting of an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.
13bp 8bp 13bp
ATAACTTCGTATA – GCATACAT -TATACGAAGTTAT
Often we found that the second G (preeceding 2 A) in the last 13 bp sequence shows an underneath peak of an A. The A signal is usually very strong while the G not much. We see this happening in several different samples and sequencing both with forward as well as the reverse primer. Overall quality of the sequence is good with no double peaks. How should I explain this?
Thank you very much for your help.
Ari

[Daniel]

I would say this is due to the secondary of your template. One of things people forget is in bigdye there is no GTP but ITP (inosine). The polymerase actually doesn’t like ITP and when faced with significant secondary structure sometime other nucleotides are inserted.

Daniel Tillett
Nucleics Support

[Anonymous]

Thank you for the quick reply.Could you suggest me how can I rule out if this is the case? Does anything added in the sequencing reaction that could help to solve a significant secondary structure?
Thank you again for your help,
Arianna

[Author]

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