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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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PCR-RFLP and Real-Time PCR Techniques in Molecular Cancer Investigations
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Biologie moléculaire et quantification : applications en hématologie
Ibtissam Chami,Dominique Bories +1 more
TL;DR: In this article, the authors discuss the use of the reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of maladie residuelle.
Identification of Aedes aegypti and its Respective Life Stages by Real-Time PCR
James C McAvin,David E. Bowles,James A. Swaby,Keith W. Blount,Jamie A. Blow,Miguel Quintana,John R. Hickman,Debra M. Niemeyer,Daniel H. Atchley +8 more
TL;DR: An Aedes aegypti specific fluorogenic probe hydrolysis (TaqMan) PCR assay was developed for real-time screening using a field-deployable thermocycler and demonstrated 100% concordance in both in vitro sensitivity and specificity.
تاداشرإ MIQE )يقيقحلا تقولا في( يمكلا لسلستلما ه رملبلا لعافت براجت شرنل تامولعلما نم نىدلأا دحلا )RT-qPCR( )لسلستلما ةرملبلا لعافتل يقيقحلا تقولا(
References
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