Hybrid amplifier

Peng H., SoellerC., CannellM. B., BowmakerG. A., Cooney R. R, andTravas-Sejdic J., Electrochemical detection of DNA hybridization amplified by nanoparticles. Biosens. Bioelectron., 21(9), 1727-1736, 2005. 

Fig. 6.148. Tolerance band within the switching stage of a hybrid amplifier during dual-state control...

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Probes that hybridize to the target and also to either preamplifier or amplifier molecules are termed label extenders. The locations of the capture and label extender probes used in the hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV-1 assays are shown in Figs. 3,4, and 5, respectively. All target probes are designed to hybridize to the most conserved regions of the genomes. For HBV, the... 

The molecular sensitivities of the first and second generations of the bDNA assays were limited by nonspecific hybridization between the amplification probes and other nucleic acids. Short regions of hybridization between any of the probes constituting the amplification system, (preamplifier, amplifier, and labeled probe) and any nontarget nucleic acid sequence leads to amplification of the background signal. Capture probes, capture extenders, and sample nucleic acid are all sources of this background hybridization (Collins et al 1997). 

In bDNA the number of target molecules is not altered. The signal of direct hybridization rather than the nucleic acid sequence itself is amplified and thus is directly proportional to the amount of target sequence present in the clinical sample. Both RNA and DNA sequences can be measured directly in clinical specimens, and there is no need to transcribe RNA into cDNA as there is with PCR. 

Figure 5.3. Systematic mating ofyeast two-hybrid bait and prey pools. Each yeast ORF was cloned individually into both as a DNA binding domain fusion (bait) and activation domain fusion (prey). The bait fusions were introduced into a MATa strain and the prey fusions were introduced into a MATa strain. The bait and prey fusions were pooled in sets of 96 clones to generate a total of 62 pools of each. The pools were systematically mated (62 x 62) in a total of 3844 crosses. Interacting clones were selected and the bait and prey inserts were PCR amplified and sequenced to determine their identify. Figure adapted from Ito et al. (2001).

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