Quantitation of non-amplified genomic DNA by bead-based hybridization and template mediated extension coupled to alkaline phosphatase signal amplification.
Klenow I polymerase activity was combined with solid phase DNA hybridization to detect non-amplified genomic DNA (gDNA) sequences from Escherichia coli. Aminopropyl-controlled pore glass surface-bound oligonucleotides were hybridized to fragmented gDNA. The template-mediated extension at the 3'-terminus of the immobilized probe was then promoted in the presence of Klenow I polymerase and digoxigenin-labeled nucleotides. Detection of the extended probes was accomplished with an anti-digoxigenin alkaline phosphatase conjugate protocol coupled to colorimetric or fluorescent detection. Using the colorimetric protocol, the proof-of-concept was established. The fluorescence-based methodology, on the other hand, provided the basis for a quantitative interpretation of the data, affording a detection limit of 5 pM gDNA.
Martins
S A M
SA
IBB-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
D000469:Alkaline Phosphatase / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics*; D017403:In Situ Hybridization / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*
D004269:DNA, Bacterial; D004247:DNA; D000469:Alkaline Phosphatase