Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
7
2.
If sufficient raw data signal is present, reduce the amount of template present in
the cycle sequencing reaction. In this case, just adding half the amount of
template may eliminate the current problem (i.e. if the original template amount
was 100 femtomoles (fmoles), use 50 fmoles).
3.
Use the preheat treatment of the template DNA prior to the cycle sequencing
reaction. The preheat treatment (see CEQ DTCS Chemistry Guide, Beckman
Coulter, Inc. P/N 718119), nicks the template DNA which alleviates the
supercoiling of the plasmid DNA. As noted above, supercoiled DNA templates
are the primary cause of current problems.
4.
Use combinations of 1, 2 and 3. Using various combinations of the first three
recommendations may also eliminate current problems. For example, use the
preheat treatment on 50 fmoles of template.
5.
Use a DNA template purification process that eliminates macromolecules such
as proteins, glycoproteins, bacterial chromosomal DNA, RNA and cell wall
debris. Most commercial DNA preparation methods yield template DNA that is
essentially free of contaminating macromolecules.
6.
Remove the template from the sequencing reaction. This is a very elaborate
process requiring special primers and reagents, but also yields sequencing
reactions that produce no current problems. The most common technique is to
use a biotinylated sequencing primer and the DynaPureII™ sequencing reaction
cleanup kit (Dynal, Inc. Oslo, Norway, product number 603.05) to purify
sequencing reaction products from contaminants such as the template DNA,
unincorporated dye-terminators, dNTPs and other salts.
An Erratic current or “Crashed Current”
In the more severe case where the current profile is erratic, the passage of dye-labeled
DNA fragments through the detection window does not pass at a constant rate and
sequencing errors are introduced. The sequencing software expects the fluorescent
molecules to be detected at a fairly constant rate, this makes predictions of the base
spacing in the base calling process. If the peak spacing is “expanded” (too few bases
per minute) as found in regions were the current has dropped quickly, the software
may insert extra bases into the sequence. Shown below are examples of erratic current
and crashed current, respectively.
