Minimizing the time required for DNA amplification by efficient heat transfer to small samples

Wittwer, Carl T.; Fillmore, G. Chris; Garling, D. J. H.

doi:10.1016/0003-2697(90)90090-v

Cited by 170 publications

(105 citation statements)

References 4 publications

Supporting

Mentioning

105

Contrasting

Order By: Relevance

“…In addition to being able to use assays based on DNA-binding dyes, hydrolysis probes and molecular beacons, it can be used for dual hybridisation probes. Up to 32 reactions are typically carried out in 5-20 µl volumes (Wittwer et al 1990) and RT-PCR reactions are completed in less than 20 min (Wittwer & Garling 1991). The Lightcycler a truly 'real-time' technique, and the fluorescence readings taken at every cycle of the PCR reaction are displayed immediately after each measurement, allowing amplification runs to be terminated or extended, as appropriate, during individual runs.…”

Section: Instrumentationmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,442

2,448

View full text Add to dashboard Cite

The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

show abstract

Section: Instrumentationmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,442

2,448

View full text Add to dashboard Cite

show abstract

“…12 Ten-minute PCR was first demonstrated in capillaries with their high surface area to volume ratio, reaction volumes of 5 to 20 l, and air thermal cycling. 13 With accurate temperature control, annealing and denaturation times of 0 second (temperature spikes) increase specificity and yield.…”

Section: Rapid-cycle Pcrmentioning

confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

The Journal of Molecular Diagnostics

Self Cite

View full text Add to dashboard Cite

From the first report of the LightCycler as a real-time polymerase chain reaction (PCR) instrument 1 clinical laboratories quickly realized its potential as a diagnostic tool. Quantitative applications important for infectious disease and oncology applications were followed by genotyping assays that took advantage of accurate temperature control to melt DNA amplicons or probe/amplicon duplexes. The first Food and Drug Administration-cleared molecular genetic assays were developed for this platform, specifically genotyping single base variants in F5 and F2. According to a College of American Pathologists survey, 2 ϳ30% of clinical laboratories report using the LightCycler for these two assays. This article will review the history of LightCycler technology, focusing on those applications widely incorporated into molecular diagnostics. In addition we will review recent developments that may impact future clinical testing.Real-time PCR instruments simultaneously amplify and detect, eliminating the need to open tubes containing PCR amplicon and therefore reducing the risk of future contamination. Fluorescent dyes or probes allow continuous monitoring as template DNA is amplified.3 By monitoring fluorescence every cycle, the amount of original target can be calculated. By monitoring fluorescence as the temperature changes, genotyping and heterozygote scanning can be performed by melting analysis, often removing the need for downstream analysis.The first two platforms developed for real-time PCR were the LightCycler (Roche, Indianapolis, IN) and the ABI 7700 (Applied Biosystems, Foster City, CA). The instruments were as different as the groups that created them. At the time, ABI was the undisputed corporate leader of PCR technology and the 7700 was a large 96-well laser-based instrument focused on throughput.

show abstract

“…Wittwer et al have shown that denaturing and renaturation are almost instantaneous (less than 1 s). 6,7 Classical kinetic studies on DNA renaturation also predict rapid annealing, because of the high primer concentration typically used in DNA amplification. 8 Therefore, PCR cycle times are ultimately limited by the elongation time and thus, the enzyme kinetics, which in turn will depend on the length of the amplified product.…”

Section: Introductionmentioning

confidence: 99%

Rapid PCR in a continuous flow device

et al. 2004

View full text Add to dashboard Cite

Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 uC) and renaturation (55 uC-68 uC) zones; above 6 mm s 21 the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from l-DNA. Amplifications at velocities ranging from 1 mm s 21 to 20 mm s 21 were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle 21) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle 21 ). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).

show abstract

Minimizing the time required for DNA amplification by efficient heat transfer to small samples

Cited by 170 publications

References 4 publications

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

LightCycler Technology in Molecular Diagnostics

Rapid PCR in a continuous flow device

Contact Info

Product

Resources

About