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Secugen - Technical Solutions - Drop Off

Sequence Appearance.

  • The DNA sequence suddenly stops or peak intensity drops off substantially. This is due to a strong secondary structure or repeats.
    Important Note: Some cloning vectors have palindromes flanking their linker and may show this drop-off effect when sequenced.

Example.

Secondary structure in plasmid sequenced under standard conditions

Secondary structure in plasmid sequenced under standard conditions

Secondary structure in plasmid sequenced under special conditions

Secondary structure in plasmid sequenced under special conditions

Possibles Causes.

  • Strong secondary structures in the DNA template. Some cloning vectors have palindromes flanking their linker and may show this drop-off effect when sequenced due to a hairpin formation.
  • Region rich in GC or GT repeats.
  • Sample contains an siRNA construct.

Treatment.

  • Apply special protocols for secondary structures o repeats rich regions.