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BioTechniques 30th Anniversary GEM Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification
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TLDR
Continuous within-cycle monitoring allows rapid optimization of amplification conditions and should be particularly useful in developing new, standardized clinical assays.Abstract:
Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR Green I, (ii) a decrease in fluorescein quenching by rhodamine after exonuclease cleavage of a dual-labeled hydrolysis probe and (iii) resonance energy transfer of fluorescein to Cy5 by adjacent hybridization probes. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy number. The sensitivity of SYBR Green I detection is limited by nonspecific product formation. Use of a single exonuclease hydrolysis probe or two adjacent hybridization probes offers increasing levels of specificity. In contrast to fluorescence measurement once per cycle, continuous monitoring throughout each cycle monitors the temperature dependence of fluorescence. The cumulative, irreversible signal of hydrolysis probes can be distinguished easily from the temperature-dependent, reversible signal of hybridization probes. By using SYBR Green I, product denaturation, annealing and extension can be followed within each cycle. Substantial product-to-product annealing occurs during later amplification cycles, suggesting that product annealing is a major cause of the plateau effect. Continuous within-cycle monitoring allows rapid optimization of amplification conditions and should be particularly useful in developing new, standardized clinical assays.read more
Citations
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References
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Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase
Randall Keichi Saiki,David H. Gelfand,Susanne Stoffel,Stephen J. Scharf,Russell Higuchi,Glenn Thomas Horn,Kary B. Mullis,Henry A. Erlich +7 more
TL;DR: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
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Molecular Beacons: Probes that Fluoresce upon Hybridization
Sanjay Tyagi,Fred Russell Kramer +1 more
TL;DR: Novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions that undergo a spontaneous conforma-tional change when they hybridize to their targets are developed.
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Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.
TL;DR: The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification.
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Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions
TL;DR: Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range.
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Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.
TL;DR: It is proposed that the larger signal in the 5' nuclease PCR assay is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR.