Section II: Troubleshooting a CEQ System Problem
6
CEQ™ 8000 Series Genetic Analysis System
Current problems
Electrokinetic Injection theory
Current problems are associated with the electrokinetic injection of sample and the
subsequent separation of the DNA sequencing fragments in the Linear Polyacrylamide
(LPA) matrix. The electrokinetic injection system will “load” negatively charged
molecules on to the LPA matrix at the tip of the capillary. These negatively charged
molecules include the dye-terminated sequencing fragments which are desirable, and
molecules like the DNA template, proteins, salts, dNTPs and the unincorporated
dye-terminators that are not desirable. Because the CEQ System uses electrokinetic
injection to load DNA sequencing fragments on to the LPA in the capillary, it is
susceptible to “overloading” problems. The most problematic of these molecules is the
DNA template from the DTCS reaction. Listed below are examples of current
problems caused by DNA templates, and to a much lesser extent proteins and other
large, charged macromolecules.
Diagnosing Current Problems (What does the current profile
look like?)
A gradually decreasing current
Supercoiled DNA templates, like plasmid DNA molecules, will load on to the LPA
matrix in the “inlet side” of the capillary and impede the flow of current through the
capillary. When the supercoiled DNA molecules load on to the LPA in the tip of the
capillaries they become “plugged” and do not allow the free flow of electrical current
through the capillary. Plugged capillaries exhibit two different current problems. In the
less severe case where the current profile shows a gradual decline without any erratic
fluctuations in the current, short reads are observed. These short reads are due to the
general slowing of the electrophoretic separation of the DNA fragments causing fewer
dye-labeled DNA fragments to pass through the detection window so fewer bases are
separated. In addition, other macromolecules that co-purify with the DNA template
can also cause current problems. These macromolecules include proteins,
glycoproteins, bacterial chromosomal DNA, RNA and cell wall debris. In general,
linear DNA molecules such as PCR products or restriction endonuclease digestion
products do not cause current problems.
Corrective actions to solve minor current problems
A gradual decrease in the current throughout the run indicates that there may only be a
slight excess of the template DNA in the sequencing reaction or the template is very
highly supercoiled. There are several solutions to this problem (listed from simplest to
most elaborate):
1.
If sufficient raw data signal is present, often just reducing the injection time is
adequate to improve the sequence call and read length. Simply rerunning the
sample and injecting for half the time may solve the problem (i.e. if the first
injection was 2kV for 15 seconds, use 2kV for 7.5 seconds).
