quantitative by running a standard curve to determine the number of target molecules in the sample.
Qp replicase has been used primarily to amplify the nucleic acid associated with infectious organisms, particularly mycobacteria,63- 64 Chlamydia,65 HIV,66 and CMV,67 but the assays are not commercially available in the United States at this time.
Signal amplification procedures differ from target amplification procedures in that the number of target sequences does not change; instead, large amounts of signal are bound to the target sequences that are present in the sample. Because the number of target sequences does not change, signal amplification procedures are inherently better at quantitating the amount of target sequences present in the clinical sample. Several signal amplification methods are available commercially.
Chiron Corp. developed and markets the branched DNA (bDNA) amplification system. The target nucleic acid for this assay can be either DNA or RNA. A series of short oligomer probes are used to capture the target nucleic acid, and additional extender probes bind to the target nucleic acid and then to multiple reporter molecules,68 loading the target nucleic acid with signal.
The bDNA signal amplification procedure is as follows. The target nucleic acid is released from the cells, the DNA is denatured if DNA is the target, and the target nucleic acid binds to capture probes that are fixed to a solid support (Fig. 7-30). Extender or preamplifier probes then bind to the captured target. The extender probes have sequences that are complementary to sequences in the target molecules as well as to sequences that are in the amplifier molecules. In the first-generation assay, the extender probes bind to a bDNA amplifier, which in turn bind multiple alkaline phosphatase-labeled nucleotides. Eight multimers or amplifiers, each with 15 branches, bind to each extender probe bound to the target. In the second- and third-generation assays, the extender probes bind preamplifiers, which in turn bind 14-15 amplifiers that can each bind to multiple alkaline phosphatase-labeled oligonucleotides (Fig. 7-31). Dioxetane is added as the substrate for the alkaline phosphatase, and chemi-
Extender probes -
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