Accelerating DNA Computing via PLP-qPCR Answer Read out to Solve Traveling Salesman Problems

An asymmetric, fully-connected 8-city traveling salesman problem (TSP) was solved by DNA computing using the ordered node pair abundance (ONPA) approach through the use of pair ligation probe quantitative real time polymerase chain reaction (PLP-qPCR). The validity of using ONPA to derive the optimal answer was confirmed by in silico computing using a reverse-engineering method to reconstruct the complete tours in the feasible answer set from the measured ONPA. The high specificity of the sequence-tagged hybridization, and ligation that results from the use of PLPs significantly increased the accuracy of answer determination in DNA computing. When combined with the high throughput efficiency of qPCR, the time required to identify the optimal answer to the TSP was reduced from days to 25 min

A review on continuous-flow microfluidic PCR in droplets : advances, challenges and future

Significant advances have been made in developing microfluidic polymerase chain reaction (PCR) devices in the last two decades. More recently, microfluidic microdroplet technology has been exploited to perform PCR in droplets because of its unique features. For example, it can prevent crossover contamination and PCR inhibition, is suitable for single-cell and single molecule analyses, and has the potential for system integration and automation. This review will therefore focus on recent developments on droplet-based continuous-flow microfluidic PCR, and the major research challenges. This paper will also discuss a new way of on-chip flow control and a rational design simulation tool, which are required to underpin fully integrated and automated droplet-based microfluidic systems. We will conclude with a scientific speculation of future autonomous scientific discoveries enabled by microfluidic microdroplet technologies

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform

Flexible sensors technology for Point-Of-Care diagnostics with integrated micro fluidics on paper

Nowadays the hospitals and the medical centres face a huge challenge finding solutions to improve the efficiency of medical diagnosis. The scope of this project was to develop a “Point-of-Care Diagnostic” (POCD) device, that can give a better alternative for genetic analysis, instead of the usual methods of PCR (polymerase chain reaction). This device is composed by three layers. The first layer which works as a transporter and filter was built on paper. The second layer is the substitute of the regular thermocycling phase in the PCR technique and the third layer incorporates an interdigital capacitor that works as a DNA (deoxyribonucleic acid) sensor with high sensitivity to detect DNA hybridisation. These last two layers were made in kapton film. The devices were produced with microfabrication methods using inkjet printing, lithographic and deposition processes. The device’s characterisation was based on impedance spectroscopy methods. With the purpose of testing the device, the capacitor was functionalised with the YWHAZ gene. However, this process can be performed with any other gene. Due to its characteristics, the device under study was designed to run RT-qPCR (Real time quantitative polymerase chain reaction) and presents itself as an effective way to substitute the traditional PCR techniques. Even more, as the transport of samples to a laboratory and the recruitment of specialised personnel are not necessary, costs and response time are reduced

Automation of cDNA Synthesis and Labelling Improves Reproducibility

Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies

AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma

A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo

Transcriptome sequencing of the Microarray Quality Control (MAQC) RNA reference samples using next generation sequencing

<p>Abstract</p> <p>Background</p> <p>Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC) reference RNA samples using Roche's 454 Genome Sequencer FLX.</p> <p>Results</p> <p>We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values ≤ 10^-20. We measured gene expression levels in the A and B samples by counting the numbers of reads that mapped to individual RefSeq genes in multiple sequencing runs to evaluate the MAQC quality metrics for reproducibility, sensitivity, specificity, and accuracy and compared the results with DNA microarrays and Quantitative RT-PCR (QRTPCR) from the MAQC studies. In addition, 88% of the reads were successfully aligned directly to the human genome using the AceView alignment programs with an average 90% sequence similarity to identify 137,899 unique exon junctions, including 22,193 new exon junctions not yet contained in the RefSeq database.</p> <p>Conclusion</p> <p>Using the MAQC metrics for evaluating the performance of gene expression platforms, the ExpressSeq results for gene expression levels showed excellent reproducibility, sensitivity, and specificity that improved systematically with increasing shotgun sequencing depth, and quantitative accuracy that was comparable to DNA microarrays and QRTPCR. In addition, a careful mapping of the reads to the genome using the AceView alignment programs shed new light on the complexity of the human transcriptome including the discovery of thousands of new splice variants.</p