Open Access
BioTechniques 30th Anniversary GEM Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification
Reads0
Chats0
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
More filters
Journal ArticleDOI
How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.
TL;DR: Recommendations for the precise estimation of PCR efficiency are proposed: one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, and using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
Journal ArticleDOI
Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.
TL;DR: Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR, and both hybridization intensity and the level of differential expression determine the likelihood of validating high-density filter array results.
Journal ArticleDOI
The External RNA Controls Consortium: a progress report.
Shawn C. Baker,Steven R. Bauer,Richard P. Beyer,James D. Brenton,Bud Bromley,John Burrill,Helen C. Causton,Michael P Conley,Rosalie K. Elespuru,Michael J. Fero,Carole Foy,James C. Fuscoe,Xiaolian Gao,David Gerhold,Patrick Gilles,Federico Goodsaid,Xu Guo,Joe Hackett,Richard D. Hockett,Pranvera Ikonomi,Rafael A. Irizarry,Ernest S. Kawasaki,Tamma Kaysser-Kranich,Kathleen F. Kerr,Gretchen L. Kiser,Walter H. Koch,Kathy Y Lee,Chunmei Liu,Z. Lewis Liu,Anne Bergstrom Lucas,Chitra Manohar,Garry Miyada,Zora Modrusan,Helen Parkes,Raj K. Puri,Laura H. Reid,Thomas B. Ryder,Marc L. Salit,Raymond R. Samaha,Uwe Scherf,Timothy J Sendera,Robert Setterquist,Leming Shi,Richard Shippy,Jesus Soriano,Elizabeth A. Wagar,Janet A. Warrington,Mickey Williams,Frederike Wilmer,Michael A Wilson,Paul K. Wolber,Xiaoning Wu,Renata Zadro +52 more
TL;DR: The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.
Journal ArticleDOI
FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.
Mark A. Poritz,Anne J. Blaschke,Carrie L. Byington,Lindsay Meyers,Kody Nilsson,David Jones,Thatcher Stephanie Anne,Thomas Charles Robbins,Beth Lingenfelter,Elizabeth A. Amiott,Amy Herbener,Judy A. Daly,Steven F. Dobrowolski,David H.-F. Teng,Kirk M. Ririe +14 more
TL;DR: An integrated diagnostic platform, the FilmArray, which fully automates the detection and identification of multiple organisms from a single sample in about one hour, and it is demonstrated that automated identification of pathogens from their corresponding target amplicon can be accomplished by analysis of the DNA melting curve of the amplicon.
Journal ArticleDOI
Real-time PCR detection chemistry
TL;DR: The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far, and comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides.
References
More filters
Journal ArticleDOI
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.
Journal ArticleDOI
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.
Journal ArticleDOI
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.
Journal ArticleDOI
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.
Journal ArticleDOI
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.