Chemiluminescent bead-based hybridization assay for the detection of genomic DNA from E. coli in purified plasmid samples.
A bead-based hybridization assay was developed for detection of traces of E. coli genomic DNA (gDNA) present in purified plasmid DNA (pDNA) samples. Standards of gDNA and pDNA samples were sheared by sonication and adsorbed onto aminopropyl controlled pore glass (CPG) particles (130 microm). A preliminary study was conducted to optimize the amount of DNA adsorbed on the particles. Results indicated that maximum attachment efficiency was obtained by adsorbing DNA for 2 h in 0.2 x SSC, pH 5.7. The DNA-bound particles were hybridized overnight with a 181-bp digoxigenin-labeled probe, specific for gDNA. Following a chemiluminescent detection protocol, signal intensities of the standards were plotted as a function of initial gDNA concentration. The calculated detection limit (LOD) was 1.4 pM of gDNA. The assay was able to detect gDNA in pure plasmid preparations at the 1% level even in the presence of 1,000-fold excess of noncomplementary target. Hybridization results were compared with a quantitative real-time PCR assay. Both methods afforded similar accurate results at the 95% confidence level.
Martins
S A M
SA
Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.
Prazeres
D M F
DM
Fonseca
L P
LP
Monteiro
G A
GA
D004247:DNA / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics*; D016678:Genome; D016680:Genome, Bacterial; D008163:Luminescent Measurements / Q000379:methods* / Q000592:standards; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods*
D004247:DNA