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Abrupt Signal Loss
Identification
- Good quality trace signal suddenly stops, or rapidly declines
- The average quality scores after the "hard stop" region are generally low
- Template has a high percentage of G and/or C nucleotides, especially in the region where the stop occurred
Causes
- Regions of strong secondary structure in the DNA template. These can fold back on themselves and form hairpin structures that the sequencing polymerase cannot pass through
- Long runs of G or C nucleotides in the template. As well as forming hairpins with strong secondary structure, they also form single stranded conformational structures that the sequencing polymerase has difficulty passing through. Long runs of guanidine are particularly problematic because of the substitution of dITP for dGTP in the BigDye sequencing mix. The BigDye sequencing DNA polymerase does not efficiently extend runs of multiple inosine residues causing the polymerase to stop in the G run regions
Overcoming Problem
- Sequence complementary strand
- Use a primer that anneals at a different position
- Incubate the reaction at 96ºC for 10 minutes before sequence cycling
- Increase the extension temperature by 2ºC to 3ºC
- Increase denaturation temperature to 98ºC
- Add DMSO to a final concentration of 5%
- Linearise the DNA with a restriction enzyme
- Shear the insert into smaller fragments (<200bp) and subclone
