Preparation of Direct, Enzyme-Labeled DNA Probes
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The direct labeling of nucleic acid probes with horseradish peroxidase (HRP) was first described by Renz at European Molecular Biology Laboratory (EMBL) in 1984 (1). The methodology was combined with enhanced chemiluminescence (2) (a light-producing HRP catalyzed reaction based on luminol) (see Chapter 26) allowing the detection of specific hybrids on membranes (3). Further development led to the availability of the first light-based nucleic acid detection system; this was capable of detecting 2.5 pg of target nucleic acid on genomic Southern blots (4). Subsequent protocol and reagent improvements now enable researchers to reliably detect 0.5 pg of target nucleic acid. Recent advances have now elucidated an analogous system for the labeling of DNA probes with alkaline phosphatase (AP), offering even higher sensitivity when combined with the new dioxetane based chemiluminescence substrates (see Chapter 26).
KeywordsBoiling Water Bath Yeast Artificial Chromosome Specific Hybrid Label Reagent Direct Label
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