Genomic DNA detection using cycling probe technology and capillary gel electrophoresis with laser-induced fluorescence.
Cycling probe technology (CPT) is an isothermal DNA analysis method that has been shown to be useful for identifying genetic markers of antibiotic-resistant bacteria in clinical settings. CPT assays have previously employed several assay methods that include polyacrylamide gel electrophoresis and magnetic beads for separations and radioisotopic and colorimetric detection for detection. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) is an alternative separation and detection method that offers attributes such as low sample consumption, short analysis times, no radiation hazards and potential for high throughput. We report on the development of a CGE-LIF CPT assay for genomic DNA from Erwinia herbicola and the comparison of this assay with a conventional 32p radioisotopic PAGE CPT assay. Separation and detection of intact and cleaved fluorescein-labeled CPT probe molecules by CGE-LIF was achieved in under 4 min through a gel-filled capillary (7 cm separation length to detector). Total time, from setup to detection, was about 1 h for the complete assay versus several hours (3-12 h) for the radioisotopic PAGE CPT assay. Similar detection limits of 10(5)-10(6) copies of genomic target DNA were observed with each assay method. This study demonstrated that CGE-LIF CPT is a suitable analysis method for the detection of genomic DNA sequences.
Dickinson Laing
Terrina
T
Canada West Biosciences Inc., Calgary, Canada.
Mah
David C W
DC
Poirier
Robert T
RT
Bekkaoui
Faouzi
F
Lee
William E
WE
Bader
Douglas E
DE
D015342:DNA Probes / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D004885:Erwinia / Q000235:genetics; D005453:Fluorescence; D016680:Genome, Bacterial; D007834:Lasers; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004269:DNA, Bacterial