Microfluidic DNA amplification - a review

The application of microfluidic devices for DNA amplification has recently been extensively studied. Here, we review the important development of microfluidic polymerase chain reaction (PCR) devices and discuss the underlying physical principles for the optimal design and operation of the device. In particular, we focus on continuous-flow microfluidic PCR on-chip, which can be readily implemented as an integrated function of a micro-total-analysis system. To overcome sample carryover contamination and surface adsorption associated with microfluidic PCR, microdroplet technology has recently been utilized to perform PCR in droplets, which can eliminate the synthesis of short chimeric products, shorten thermal-cycling time, and offers great potential for single DNA molecule and single-cell amplification. The work on chip-based PCR in droplets is highlighted

Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Handling the Microbial Complexity Associated to Ticks

Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. In the last years, high-throughput detection and sequencing technologies (HTT) have revealed that individual ticks carry a high diversity of microorganisms, including pathogenic and non-pathogenic bacteria. Despite several studies have contributed to the availability of a catalog of microorganisms associated to different tick species, major limitations and challenges remain ahead HTT studies to acquire further insights on the microbial complexity associated to ticks. Currently, using next generation sequencing (NGS), bacteria genera (or higher taxonomic levels) can be recorded; however, species identification remains problematic which in turn affects pathogen detection using NGS. Microfluidic PCR, a high-throughput detection technology, can detect up to 96 different pathogen species, and its combination with NGS might render interesting insights into pathogen-microbiota co-occurrence patterns. Microfluidic PCR, however, is also limited because detection of pathogen strains has not been implemented, and therefore, putative associations among bacterial genotypes are currently unknown. Combining NGS and microfluidic PCR data may prove challenging. Here, we review the impact of some HTT applied to tick microbiology research and propose network analysis as an integrative data analysis benchmark to unravel the structure and significance of microbial communities associated to ticks in different ecosystems

Development of DNA assembly and error correction protocols for a digital microfluidic device

Customized production of synthetic DNA from oligonucleotides is in high demand. However, current technologies are costly and labor-intensive. A microfluidic technology can significantly decrease cost and labor. The purpose of this study was to develop a gene assembly protocol that was utilized on the Mondrian™ SP digital microfluidic device. The fragment of the human influenza virus hemagglutinin (HA) gene (339 bp) was assembled from 12 oligonucleotides by the Gibson assembly method and error corrected with CorrectASE™ enzyme twice. The samples were analyzed by Sanger sequencing to verify the final accuracy of the assembly. A complete automation of droplet generation and movement on digital microfluidic droplet technology was achieved in the study. The reactions were scaled down to 0.6-1.2 µL. Gibson assembly, PCR, and enzymatic error correction reactions were optimized and combined in a single protocol. The microfluidic assembly demonstrated approximately 3 errors/kb error frequency. Polymerase chain reaction supplemented with additional MgCl2, Phusion, and PEG 8000 provided amplification of the assembly and error correction products. The lowest error frequency of 0.3 errors/kb was achieved after one CorrectASE™ treatment. However, microfluidic error correction was not reliable due to CorrectASE™ interactions with the microfluidic surface, which need to be the subject of future work

A multi-function, disposable, microfluidic module for mutation detection

Recognition of point mutations in a codon 12 of the K-ras gene, most frequently observed, is considered to be useful in the early diagnosis of several types of the human cancers. We have developed a multifunction, disposable, microfluidic module which detects low-abundant point mutations in human genomic DNA in modular architecture. Each functional component including a microfluidic PCR reactor, a passive diffusional micromixer reactor, and a microfluidic LDR reactor was separately designed and fabricated. Fluidic interconnects were also developed to make a fluidic passage between the functional components. Polycarbonate substrates were micro-molded, using hot embossing with micro-milled brass mold inserts to make all microfluidic components. Developed microassembly using passive alignment features, fabricated on all components, was used to assemble the functional components with the fluidic interconnects using an adhesive bonding technique. Thermal simulations were employed to ensure uniform thermal distributions in the microfluidic PCR and LDR reactors, to isolate the mixing junction in order to avoid heat–induced bubble formation in the passive micromixer reactor, and to have minimal thermal crosstalk due to the asymmetric thermal zones in the PCR and the LDR reactors. A control system was developed to control temperatures enabling thermal cycling in the microfluidic PCR and LDR reactor. LDR products were produced using the module within an hour with DNA sample, which had the ratio of 1:200. Total reaction time was about 67 minutes. By applying an enzyme as a purification of PCR products, a LDR analysis can be optimized and minimized to reduce the false positive signals and inconstant results generated by PCR products during the LDR. The purification system allowed us to successfully quantify the amount of mutant alleles in the genomic DNA. The high degree of accuracy in this module can also facilitate the detection of low-frequency point mutation occurred in other functional genes. This module, fabricated using replication technologies of polymers will be able to supply low cost, disposable detection tools for known disease-causing mutations and also expand to other PCR-based detection assays in diagnostic applications

Microfluidic polymerase chain reaction

We implement microfluidic technology to miniaturize a thermal cycling system for amplifying DNA fragments. By using a microfluidic thermal heat exchanger to cool a Peltier junction, we have demonstrated rapid heating and cooling of small volumes of solution. We use a miniature K-type thermocouple to provide a means for in situ sensing of the temperature inside the microrefrigeration system. By combining the thermocouple, two power supplies controlled by a relay system, and computer automation, we reproduce the function of a commercial polymerase chain reaction thermal cycler and demonstrate amplification of a DNA sample of about 1000 base pairs

Practical considerations for plant phylogenomics

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143756/1/aps31038_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143756/2/aps31038.pd