For problems associated with sequencing:

Primer related

1. Poor priming resulting in very weak signal
   • Melting temperature too low: low G/C content or short primer
   • Secondary structure of primer, particularly at the 3' end
   • Secondary structure of template in region of hybridization
2. Adequate signal strength but data is noisy
   • Secondary hybridization site; generally will see too many peaks (peaks under peaks). More stringent annealing conditions can eliminate this.
   • Impure primer; may see a "shadow sequence"
3. Incorrect primer sequence (for custom primers)
   • Mismatch on 3' end will generally result in completely flat line or no signal

Template related

1. Poor template quality
   • Impurities such as protein, RNA, and salts in template DNA will produce high background, and poor quality data. Additionally, some peaks will have fairly large signal underneath the correct signal
2. Amount of template DNA (with optimal quality DNA)
   • Not enough template: low signals, noisy data, errors and ambiguities are high
   • Too much template: Peaks in sequencing data show trailing and may be accompanied by a weaker signal
3. G/C rich template (particularly when % GC exceeds 62%) are more difficult to sequence
   • Overall weak signal resulting in noisy data: presumably this is related to the tighter duplex and greater difficulty of denaturing the template and keeping it denatured, as well as the presence of secondary structure
4. Unbalanced template base composition results in high baseline noise in one or more colors
   • With A/T rich template, some additional G and C noise may be observed throughout
   • Similarly, A and T noise may appear in G/C rich template
5. Template secondary structure, inverted repeats for example
   • Good signals in the beginning but signals drops off suddenly

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