Quantitation of non-amplified genomic DNA by bead-based hybridization and template mediated extension coupled to alkaline phosphatase signal amplification.

ABSTRACT

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.