The cePRO 9600™ system fromCombiSep provides high-throughput
automated separation and quantitation of DNA fragments by 96-capillary
array gel electrophoresis. On-line UV detection provides a sensitive,
cost-effective means of analysis and eliminates the need for DNA
labeling chemistries. The method provides superior separation
resolution, improved quantitation and increased automation and
throughput compared to agarose or polyacrylamide slab gels.
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Introduction DNA fragment analysis
is widely employed for optimization and quality control of polymerase
chain reactions (PCR) and in genotyping studies. Agarose or
polyacrylamide slab gel electrophoresis remains a common technique for
DNA analysis despite time-consuming gel preparation and sample loading,
exposure to toxic reagents and semi-quantitative data analysis.
Capillary gel electrophoresis (CGE) increasingly has emerged as a
technique for performing DNA analysis, due to its high separation
efficiency and resolving power, low sample requirements, on-line
detection and automated operation.
The cePRO 9600™ system is a 96-capillary array electrophoresis
instrument utilizing on-line ultraviolet (UV) absorbance detection. As
DNA possesses high absorptivity at 254 nm, it is possible to directly
analyze DNA without the use of expensive, toxic labels commonly employed
in fluorescence-based techniques. The cePRO 9600™ is capable of
analyzing 96 DNA samples in approximately 1 h, providing a
high-throughput and cost-effective means for DNA analysis.
Experimental Conditions A
proprietary low-viscosity, replaceable gel sieving matrix was developed
in-house for DNA separations. DNA ladders were purchased from Fermentas
Inc. (Hanover, MD, USA) or New England Biolabs (Beverly, MA, USA).
Separations were performed on a cePRO 9600™ system (CombiSep Inc.,
Ames, IA, USA) controlled by CombiSep's cePRO Manager®
software package. Data analysis was facilitated by CombiSep DNA Size™
software.
Figure 1. A) A 96-capillary
separation of an 100-bp ladder (Fermentas) with added
200-bp/2000-bp sizing markers displayed as a
"gel-like" image. Migration times were normalized to
the sizing markers. The total sample concentration was 10 ng/L.
CGE conditions: -5 kV, 50 sec injection; -9 kV separation. The
corresponding electropherogram for capillary #1 is shown to the
left of the graph.
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Results Figure 1A depicts the
simultaneous separations of 96 individual 100-bp DNA ladder samples. The
separations were normalized using co-injected DNA markers at 200 bp and
2000 bp. The ladder from capillary #1 was used to construct a
calibration curve of migration time versus DNA size. Predicted DNA
fragment sizes in the remaining 95 capillaries were accurate to 2.2% or
less with high reproducibility (RSD ≤ 1%).
