Amplification systems

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). 

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

PCR and related target amplification systems typically employ a single pair of primers. Each primer is usually 20 to 40 bases in length and anneals to the complementary sequence on the target nucleic acid to initiate the amplification reac-... 

In summary, bDNA has a number of distinct theoretical and practical advantages over target amplification systems for direct quantitation of specific nucleic acid sequences. The following sections review the specific clinical and research applications of this technology. 

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. 

Fahey, E., Kwoh, D. Y. and Gingeras, T. R. Self-sustained sequence replication (3SR) an isothermal transcription-based amplification system alternative to PCR , PCR Methods Appl., 1, 25-33 (1991). 

An amplification reaction that is used to amplify target RNA or denatured DNA is called the transcription-based amplification system (TAS). This technique involves using an enzyme called reverse transcriptase and a primer with sequence complementary to the sample target RNA molecule in order to synthesize a complementary DNA (cDNA) copy of the sample target RNA. After denaturation to separate the strands, another primer and additional reverse transcriptase are added to synthesize a double-stranded cDNA molecule. Since the first primer has also an RNA polymerase binding site, it can, in the presence of T7 RNA polymerase, amplify the double-stranded cDNA to produce 10 to 100 copies of RNA. The cycle of denaturation, synthesis of cDNA, and amplification to produce multiple RNA copies is repeated. With as few as four cycles, a 2- to 5-millionfold amplification of the original sample RNA target is possible. However, the time required to achieve a millionfold amplification is approximately 4 hours, which is the same amount of time required by PCR. The TAS requires, however, the addition of two enzymes at each cycle and, as such, can be cumbersome. 

An amplification system that actually amplifies exponentially RNA probe sequences bound to the target sequence, in contrast to PCR and TAS systems, which amplify target sequences, is the Q-beta replicase system (B4). Although this system can achieve a million- to billionfold amplification in 15 minutes at 37°C, background signal due to nonhybridized probes is reported to be very high. 

An amplification system that has been successfully commercialized is called the ligase chain reaction (B4). The basis for this reaction lies in using two small single-stranded DNA probes of 10 to 20 bases in length to anneal to target DNA and an enzyme called DNA ligase to link the two probes. Twenty to 50 cycles of this reaction can yield sufficient amplified product. 

The next key point is to realize that each enzyme in the pathway exists in both active and inactive forms. cAMP initiates a cascade of reactions by activating protein kinase A (PK-A)," the active form of which activates the next enzyme in the sequence, and so on. At the end of the day, glycogen phosphorylase is activated and glucose or ATP is produced. This signaling pathway is a marvelous amplification system. A few molecules of glucagon or adrenaline may induce formation of many molecules of cAMP, which may activate many of PK-A, and so on. The catalytic power of enzymes is magnified in cascades of this sort. 

Castelain, S., V. Descamps, V. Thibault, C. Francois, et al. TaqMan Amplification System with an Internal Positive Control for HCV RNA Quantitation. Journal of Clinical Virology 31 no. 3 (2004) 227-234. 

Mori, M., Fujihara, N., Mise, K., and Furusawa, I. (2001). Indncible high-level mRNA amplification system by viral replicase in transgenic plants. Plant J. 27(1) 78-86. 

Prior to deconvolution, the convolved spectrum was corrupted with additive noise to form rms signal-to-noise ratios of oo 1, 580 1,115 1, 60 1, and 30 1 for traces (c)-(g), respectively. The noise generated is similar to white noise (or detector noise) after passing through an amplification system with a negligible time constant. A typical spectrum would be recorded with the higher noise frequencies already attenuated, and thus our example may not represent a realistic situation. It should also be pointed out that the simulated noise differs from trace to trace in Fig. 3 only in amplitude and is thus not truly random. 

Fig. 30. Detection of mRNA on a membrane or in situ with labeled gene probes. A Detection of mRNA with a fluorescein-labeled single stranded nucleic acid probe, using POD-conjugated anti-fluorescein antibody. B Use of two gene probes labeled with different molecules (fluorescein and digoxigenin) and detected with specific antibodies, both coupled to AP and using two substrates, leading to differently colored products. This in situ hybridization scheme allows the simultaneous detection of two mRNA species in a tissue or cell preparation. C Amplification systems involving more than one antibody can be used to increase specificity and signal intensity.

Apart from concentrating the analyte prior to immunoassay, several other possibilities exist for enhancing the sensitivity of the procedure. These constitute introduction of various amplification systems, such as the peroxidase-antiperoxidase system (110), the streptavidine-biotine system (111, 112), and the substrate amplification system (113, 114), which favor the enhancement of the signal per ligand. 

PHYSICAL DEVELOPMENT AND OTHER IMAGE AMPLIFICATION SYSTEMS 113... 

Latent images or faint images in silver metal or other materials can be amplified by redox chemistries other than metal deposition. Several dye-forming redox chemistries have been discovered in which metal complexes serve as catalysts, catalyst precursors or one of the redox partners. The applications of coordination compounds in physical development and image amplification systems are therefore quite broad and diverse. 

In another interesting amplification system, an image in silver or other noble metal nuclei is used for catalysis, and a cobalt(III) complex is used as an oxidant in place of a peroxy compound. The amplification permits less silver to be used. An imagewise distribution of silver nuclei is associated with a non-diffusible dye-forming coupler, and the image is amplified by (1) imbibing the system with a solution containing a color developer such as a p-phenylenediamine and (2)... 

---