Secugen - Failed DNA Sequencing Reaction
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Secugen - Technical Solutions - Failed DNA Sequencing Reaction or “Dirty” Sequence

Sequence Appearance.

  • Electrophoregram has mixed peaks or is mostly blank.
  • Many ‘N’s in the sequence, if bases are called at all.
  • The read sequence file yields a no recognizable sequence.
  • Raw data has signal intensity in the low hundreds.

Example.

No Reaction


Failed DNA Sequencing Reaction


Possibles Causes.
The causes for these kind of results can be several, the most common are the following ones:

  • DNA Template concentration is too low.
    Important Note: measuring DNA concentration by UV absorption is often inaccurate and concentration is frequently overestimated. Agarose gels or espectrofluotometry are a much better method to estimate quality and quantity of DNA samples. In this case the Raw Data signal is low, around 100, due to poor DNA sequencing reaction.
  • No correct primer was added to the reaction, so binding between the DNA and the primer cannot occur. No DNA sequencing reaction, and hence Raw Data signal usually below 100 units.
  • Template purification is not working properly. Sometimes the sample preparation is contaminated with ethanol or other molecules that inhibit the DNA Polymerase, yielding no sequencing reaction.

Treatment.

  • Check the concentration of the template by agarose gel or espectrofluorometry to ensure that it is in the ranges described in the Sample Submission Guidelines.
  • Check the primer sequence against the template sequence to ensure that there is a proper binding site.
  • Improve the quality of the template preparation, mainly for the primers making fresh dilution each time in case problems were observed.