Section II: Troubleshooting a CEQ System Problem
14
CEQ™ 8000 Series Genetic Analysis System
4.
If the correct amount of template was added and the preheat treatment does not
yield a substantial increase in Raw Data signal increase the number of cycles in
the thermal cycling program from 30 to 40 or 50.
5.
If the correct amount of template was added and the Preheat Treatment and / or
increasing cycle number does not yield a substantial increase in Raw Data signal,
a DNA Polymerase inhibitor may be present (do not resuspend DNA in DEPC
treated water). In this situation further purification of the DNA template may be
required. In some cases a simple ethanol precipitation of the plasmid will remove
the inhibitor, whereas other situations may require the use of commercial DNA
preparation methods such as the Qiagen QiaQuick kit.
Low Raw Data Signal Due to Primer Quantity and Quality Problems
The amount and purity of sequencing primers can also cause low raw data signal. For
example, having insufficient quantities of the primer or having a poor quality primer
(e.g. a primer that has not been totally deprotected after synthesis) will yield low Raw
Data signal. Many suppliers of oligonucleotides provide sufficiently pure preparations
of primers. Most of these vendors will provide “desalted primers” which function well
for DNA sequencing. However, further purification of the primers by TSP (Trityl
Specific Purification) or HPLC (High Performance Liquid Chromatography) is highly
recommended when the highest quality DNA sequencing results are desired. Also,
primers purified by denaturing gel electrophoresis have been shown to contain
inhibitors of the DNA Polymerase and therefore, are not recommend for use in DNA
sequencing reactions.
Primer Design Problems that Cause Low Raw Data Signal
Another cause of primer related low raw data signal is poor primer design. Primers
should be designed so that they have reasonably high T
m
’s (T
m
greater than 55°C), no
internal secondary structures (such as hairpin loops) and a low probability of forming
“primer dimers” (the 3' ends of the primers binding to each other and are extended).
The T
m
of the primer must be at least 5°C higher than the annealing temperature of the
cycle sequencing program (50°C) to adequately bind to the template DNA and be
extended by the polymerase. Internal secondary structures and primer dimers are
thermodynamically favored over primer- template binding so they reduce the effective
concentration of the primer in the sequencing reaction.
