Section II: Troubleshooting a CEQ System Problem
12
CEQ™ 8000 Series Genetic Analysis System
Raw Signal Problems
Types of Raw data signal problems
There are several types of raw data signal problems. The most common problem is
signal levels that are too low. In this case there is insufficient fluorescence signal
(shown as Activity (cnts) on the electropherogram) to generate an accurate base call.
Another possible problem is when there is too much raw data signal, which causes the
photomultiplier detection system to be saturated. This is caused by too many
fluorescently labeled DNA sequencing fragments. Another category of raw data signal
problem is too many fluorescent peaks in the electropherogram. This can be caused by
two independent sequencing events present in the same sample. This phenomenon is
caused by either having two or more templates in the reaction or multiple (2 or more)
primer binding sites on a DNA template. A variation on the previous problem is
referred to as a “pre-peak problem”. Prepeaks are caused by poor quality primer
synthesis where a high proportion of n-1 primer (primers which are one base short of
the desired length) are present. The previously mentioned problems, as well as several
other possible problems, will be discussed in more detail in the following sections.
Diagnosing signal level problems
The first step in diagnosing a Raw Data signal problem is to look at the fluorescent
signal (Raw Data signal) in the Sequencing Analysis application. The data, which is
plotted as Activity (cnts) versus Time (Minutes), should show fluorescent signal
(Activity cnts) in the range of 5,000–125,000 cnts. The signal will be high in the
beginning and gradually decline throughout the run.
Causes of Low Raw Data Signal
Low Raw Data Signal due to not enough template
Low raw data signal can be caused by a variety of issues. One of the most common
causes is lack of sufficient DNA template in the cycle sequencing reaction. It is vital to
the success of CE sequencing to have the correct amount of template in the reaction. It
is recommended that 50–100 fmoles of plasmid DNA be used in the cycle sequencing
reaction. This provides enough template to generate an adequate amount of
fluorescently labeled DNA sequencing fragments yet not so much as to cause current
problems (see “Current problems” on page 6). Half this amount of DNA template
(25–50 fmoles) should be used for single stranded DNA templates such as M13 phage
DNA and even less DNA is needed for small PCR products (10–50 fmoles for PCR
products less than 3KB in length). In many cases the amount of template added to the
reaction is not determined and therefore, insufficient template is present. In other
cases, an incorrect approximation of the DNA concentration is made.
Spectrophotometric estimation of DNA samples is only valid if the DNA is pure (as in
the case of commercial DNA template purification methods). Crude preparation of
DNA templates which have substantial amounts of protein and/or RNA will over
estimate the concentration of the template and cause the user to add too little DNA to
the sequencing reaction (as in the case of crude alkaline lysis minipreps).
