Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment

Strohmeier, Oliver; Laßmann, Silke; Riedel, Bianca; Mark, Daniel; Roth, Günter; Werner, Martin; Zengerle, Roland; Stetten, Felix von

doi:10.1007/s00604-013-1099-z

Cited by 23 publications

(21 citation statements)

References 25 publications

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“…2D). 67,68 Airdried reagents for the PCR reaction are dissolved by the sample inside of the chambers. Thermocycling is conducted in a commercial thermocycler.…”

Section: Centrifugal Microfluidic Devicesmentioning

confidence: 99%

Polymerase chain reaction in microfluidic devices

2016

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The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection. The development of isothermal nucleic acid amplification and digital PCR microfluidic devices within the last five years is also highlighted. Furthermore, we introduce various commercial microfluidic PCR devices.

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“…2D). 67,68 Airdried reagents for the PCR reaction are dissolved by the sample inside of the chambers. Thermocycling is conducted in a commercial thermocycler.…”

Section: Centrifugal Microfluidic Devicesmentioning

confidence: 99%

Polymerase chain reaction in microfluidic devices

2016

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“…number of experimental works demonstrate this application, [61][62][63][64][65] few incorporate a computational analysis. Naghdloo et al worked on the simulation of an LOD device proposed for nested-polymerase chain reaction (PCR) using a combination of convective and radiative heat transfer mechanisms.…”

Section: Lab On a Chip Critical Reviewmentioning

confidence: 99%

“…The general governing equation for a solid domain is eqn (61), which is related to the conservation of momentum. 127 Fig.…”

Section: Solids Computational Analysismentioning

confidence: 99%

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Mathematical modeling and computational analysis of centrifugal microfluidic platforms: a review

et al. 2020

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“…The assay was previously demonstrated as a conventional nested PCR, consisting of a universal pre-amplification and animal-group-specific main-amplification of two mitochondrial gene parts [ 11 ]. Here, adaptions and extensions of the assay allow a full integration of all required reagents into the Microfluidic App, which augments the RGQ to perform all liquid handling steps for the nested PCR protocol utilizing tailored centrifugal microfluidics instead of modifying the Rotor-Gene [ 32 – 35 ]. In turn, identification of twelve different animal groups (Equidae, Phasianidae, Felidae, Cervidae, Canidae, Mustelidae, Leporidae, Caprinae, Bovini, Sus scrofa , human and rodents (mouse and squirrel)) including all required controls was automatically performed to examine sensitivity, specificity and fluidic reliability of the system.…”

Section: Introductionmentioning

confidence: 99%

Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment

et al. 2015

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Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

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Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment

Cited by 23 publications

References 25 publications

Polymerase chain reaction in microfluidic devices

Polymerase chain reaction in microfluidic devices

Mathematical modeling and computational analysis of centrifugal microfluidic platforms: a review

Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment

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