Principles Of bDna Technology

The basic principles of bDNA technology will be described here taking the commercially available VERSANTâ„¢ assays (Bayer Diagnostics, Berkeley, CA) for determination of viral loads as an example. After release of genomic DNA or RNA by a lysis reagent, capture of the target nucleic acids to the bottom of a microtiter plate well is mediated by probes termed capture extenders (Fig. 1). Then, a second series of target-specific probes hybridize to various sites of the captured DNA or RNA. These label extenders are designed in such a way that two of them must be bound to adjacent regions of the target to enable efficient hybridization of the preamplifier molecule. The resultant cruciform label extender-preamplifier complex and the incorporation of nonnatural, synthetic nucleotides (i.e., iso-C and iso-G) into selected target and amplification probes significantly reduce the assay background by minimizing nonspecific probe interactions. The bDNA amplifiers as the key element of the whole technology finally hybridize to the preamplifiers. Every bDNA molecule has 15 arms, and three sites for hybridization of alkaline phosphatase-labeled probes are located on each of the branches resulting in a tremendous enhancement of the generated signal. The enzyme catalyzes the dephos-phorylation of the chemiluminescent substrate Lumi-Phos PlusTM, and the intensity of the light emission is directly proportional to the original amount of target sequence present in the sample. Quantitation is possible by a standard curve defined from preparations containing known concentrations of recombinant DNA.[1-4]

bDNA technology, in principle, can be used for detection and quantitation of every known nucleic acid sequence. The specificity and sensitivity of the methodology, however, largely depend on the judicious design of the probe sets. This task is partly facilitated by a commercial software application (ProbeDesignerTM), which allows for the recognition of unwanted nonspecific hybridization events and provides an algorithm for the simultaneous design of probe sets for multiple targets.[5] Because bDNA amplification is a nonenzymatic process that does not change the original amount of target nucleic acid sequences, this technique, in comparison to enzy-matically mediated amplification systems, is less prone to contamination, sample-to-sample variation, and the influence of inhibitory substances contained in clinical specimens. On the other hand, target amplification in general still has greater analytical sensitivity than bDNA and provides greater feasibility in design when novel targets are approached because only one appropriate pair of primers and not a whole set of hybridization probes is needed.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment