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′-term...

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Bibliographic Details
Published in:Biotechnology letters 2010-02, Vol.32 (2), p.229-234
Main Authors: Martins, S. A. M, Prazeres, D. M. F, Fonseca, L. P, Monteiro, G. A
Format: Article
Language:English
Subjects:
Alkaline phosphatase
Alkaline Phosphatase - genetics
Applied Microbiology
Biochemistry
Biological and medical sciences
Biomedical and Life Sciences
Biotechnology
Colorimetry
Conjugates
Deoxyribonucleic acid
DNA
DNA - analysis
DNA - genetics
DNA, Bacterial - genetics
E coli
Escherichia coli
Fundamental and applied biological sciences. Psychology
Genome, Bacterial - genetics
Genomics
Hybridization
In Situ Hybridization - methods
Life Sciences
Microbiology
Nucleic Acid Amplification Techniques - methods
Nucleotides
Original Research Paper
Polymerase
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Summary: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.
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-009-0149-4