Signal Amplification

Summary

Signal amplification methods were initially designed as an alternative to target amplification technologies, such as polymerase chain reaction (PCR), to minimize the possibility of contamination by target amplification products. Unlike target amplification, signal amplification methods (as defined herein) do not rely on enzymes for amplification. Probe-based amplification techniques, such as cleavage-based amplification and rolling circle amplification, are enzyme-based and will not be covered in this chapter. Signal amplification enhances or amplifies the signal generated from the probe molecule hybridized to the target nucleic acid sequence. Advantages of signal amplification methods include specific detection, dynamic range, ease of use, and reproducibility.

To date, these methods have been challenged by advanced or automated target amplification methods such as real-time PCR. Signal amplification technologies include hybrid capture (HC) and branched DNA (bDNA) assays. The HC method was initially developed and marketed by Digene Corporation (Gaithersburg, MD) which was acquired by Qiagen (Valencia, CA) in 2007. The bDNA method was initially developed by Chiron (Emeryville, CA), marketed by Bayer Diagnostics (Emeryville, CA). CA) CA), whose diagnostic division was acquired by Siemens (Tarrytown, NY) in 2006. Due to the increasing demand for fast detection times, automation, and multiplexing, the popularity of commercial signal amplification methods has declined in recent years. clinical virology laboratories.

Methods

  • General information

T7 RNA polymerase, FastAP thermosensitive alkaline phosphatase (FastAP), T4 PNK, Bsm DNA polymerase, Klenow fragment exopolymerase and Nb.Bpu10I cleavage endonuclease was purchased from MBI Fermentas. NTP and dNTP were purchased from TransGen Biotech (Beijing, China). [γ-32P]ATP, [α-32P]dATP and [α-32P]dCTP were purchased from Furui Biological Engineering (Beijing, China). Hemin was purchased from Alfa Aesar, while the stock solution was made up of dimethylsulfoxide and stored in the dark at -20 °C. ABTS was purchased from Wolsen (Xian, China); H2O2 was purchased from Bodi Chemical Holding Co., Ltd (Tianjin, China); and all oligonucleotides were purchased from Sangon Biotech (Shanghai, China) and purified by HPLC.

  • RNA transcript

RNA substrates were synthesized by RNA transcription using T7 RNA polymerase and a double-stranded DNA template having the following sense sequence 5′-GAATT CTAATACGACTCACTATA-RNA-3′ (italics indicate the promoter sequence of the T7 RNA polymerase). A transcription reaction containing 0.5 μM template DNA, 10 mM NTP, 1 × T7 RNA polymerase buffer (40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 10 mM dithiothreitol, 6 mM MgCl2, 2 mM spermidine) and 1.25 unit per μl of RNA polymerase were incubated at 37°C for 4 h, followed by ethanol precipitation and PAGE purification.

  • Labelling reaction

A reaction mixture containing oligonucleotides was incubated with 50 mM Tris-HCl (pH 7.8), 40 mM NaCl, 10 mM MgCl2, 1 mg ml−1 BSA, 10 μCi [γ-32P]ATP, and 10 units PNK for 1 h at 37 °C for DNA phosphorylation. The labelled product was purified by 10% denaturing PAGE. Single-stranded DNA could be labelled directly, but RNA substrates obtained by in vitro transcription must be treated with alkaline phosphatase as follows: RNA was incubated with 0.05 unit per μl FastAP in 10 mM Tris–HCl (pH 8, 0), 5 mM MgCl2, 100 mM KCl, 0.02% Triton X-100 and 0.1 mg ml−1 BSA at 37 °C for 10 min, and then extracted with phenol-chloroform and recovered by precipitation with ethanol.

  • Kinetic analysis of DNAzymes

The cleavage reaction containing 20 μM DNAzymes, 100 nM 32P-labeled Flag-RNA, 10 mM Tris-HCl (pH 8.5), 100 mM KCl, 10 mM MgCl2, and 0.01 mg ml-1 BSA was incubated at 37 °C. The reaction was stopped after a designated period of time, by the addition of EDTA (pH 8.0) to a final concentration of 30 mM. Cleavage products were separated by 10% denaturing PAGE and quantified by phosphorimager (PerkinElmer Cyclone Plus Storage Phosphor System). Time courses for each deoxyribozyme were run at least twice, using more than eight-time points for each. Experimental data were fitted to the exponential equation Y=Ymax(1-e(−kobs)) using nonlinear regression analysis in GraphPad Prism 4, from which the observed rate constant (kobs) and yield were determined. division maximum (Ymax).

  • RNA cleavage and extension

A cleavage reaction mixture containing 6 µM DNAzymes, 150 nM 5′-32P-labeled Flag-RNA, 10 mM Tris-HCl (pH 8.5), 100 mM KCl, 10 mM MgCl2 and 0.01 BSA was incubated. mg ml−1 at 37°C. C for 1 h. Subsequently, PNK (0.5 unit per μl) and Bsm polymerase (0.4 unit per μl) was added to the reaction mixture for 30 min. All reactions were analyzed by running a 10% denaturing PAGE.

  • Colourimetric detection

Flag-RNA targets were first added to a detection solution to a final volume of 10 μl, containing target-specific concentrations, 0.25 μM fat, 0.25 unit per μl PNK, 10 mM Tris-HCl (pH 8.5), 100 mM KCl, 10 mM MgCl2 and 0.1 mg ml−1 BSA, and incubated at 37 °C for 150 min. Then, 0.25 μM Ta, dNTP (250 μM), Nb.Bpu10I (0.125 unit per μl) and Klenow exopolymerase (0.125 unit per μ1) was added to a final volume of 20 μl and incubated for 60 min at 37 °C. c. . Finally, 1.7 μl of Tris-HCl (pH 7.3), hemin (1.8 μM), H2O2 (2.1 mM) and ABTS (2.1 mM) were added to the reaction mixture at 25 °C. C and the colourimetric result was immediately recorded with a digital camera.

  • Real-time fluorescence assay

Real-time fluorescence assay was performed in a 20 μl reaction mixture containing 10 mM Tris-HCl (pH 7.0), 100 mM KCl, 10 mM MgCl2, 0.1 mg ml−1 BSA , dNTP (250 μM), 0.17 unit per μl Nb.Bpu10I, 0.17 unit per μl Bsm DNA polymerase, 0.08 unit per μl PNK, malachite green (15 μmol), 0.25 μM fCat, 0.25 μM Tb and specific concentrations of Flag-RNA targets. The reaction was performed in a real-time PCR system (channel 4, excitation 600–640nm, emission 666–740nm; PikoREAL, Thermo, USA) at 39 °C. Real-time fluorescence intensity was monitored at 1 min intervals.