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

Labeling of Unique Sequences in Double-Stranded DNA at Sites of Vicinal Nicks Generated by Nicking Endonucleases

We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.Wallace A. Coulter Foundatio

Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes.

With defined culture protocol, human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for cardiac disease therapies. In this study, we successfully generated a highly pure population of human cardiomyocytes (hCMs) (>95% cTnT(+)) from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types

Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases

Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila

Mitochondrial DNA (mtDNA) often exists in a state of heteroplasmy, in which mutant mtDNA co-exists in cells with wild-type mtDNA. High frequencies of pathogenic mtDNA result in maternally inherited diseases; maternally and somatically acquired mutations also accumulate over time and contribute to diseases of ageing. Reducing heteroplasmy is therefore a therapeutic goal and in vivo models in post-mitotic tissues are needed to facilitate these studies. Here we describe a transgene-based model of a heteroplasmic lethal mtDNA deletion (mtDNA^Δ) in adult Drosophila muscle. Stimulation of autophagy, activation of the PINK1/parkin pathway or decreased levels of mitofusin result in a selective decrease in mtDNA^Δ. Decreased levels of mitofusin and increased levels of ATPIF1, an inhibitor of ATP synthase reversal-dependent mitochondrial repolarization, result in a further decrease in mtDNA^Δ levels. These results show that an adult post-mitotic tissue can be cleansed of a deleterious genome, suggesting that therapeutic removal of mutant mtDNA can be achieved

Nano-enabled bioanalytical approaches to ultrasensitive detection of low abundance single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) constitute the most common types of genetic variations in the human genome. A number of SNPs have been linked to the development of life threatening diseases including cancer, cardiovascular diseases and neurodegenerative diseases. The ability for ultrasensitive and accurate detection of low abundant disease-related SNPs in bodily fluids (e.g. blood, serum, etc.) holds a significant value in the development of non-invasive future biodiagnostic tools. Over the past two decades, nanomaterials have been utilized in a myriad of biosensing applications due to their ability of detecting extremely low quantities of biologically important biomarkers with high sensitivity and accuracy. Of particular interest is the application of such technologies in the detection of SNPs. The use of various nanomaterials, coupled with different powerful signal amplification strategies, has paved the way for a new generation of ultrasensitive SNP biodiagnostic assays. Over the past few years, several ultrasensitive SNP biosensors capable of detecting specific targets down to the ultra-low regimes (ca. aM and below) and therefore holding great promises for early clinical diagnosis of diseases have been developed. This mini review will highlight some of the most recent, significant advances in nanomaterial-based ultrasensitive SNP sensing technologies capable of detecting specific targets on the attomolar (10-18 M) regime or below. In particular, the design of novel, powerful signal amplification strategies that hold the key to the ultrasensitivity is highlighted

A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide

This is the final version of the article. Available from the publisher via the DOI in this record.Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA-protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ~18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA-protein interactions.Netherlands Organisation for Scientific Research and the Netherlands Institute for Space Research [ALW-GO-PL/ 08-08]; NWO Vidi grant [864.11.005 to S.J.J.B.]. Funding for open access charge: Microbiology department/ Wageningen UR library

CSI ook in de Plantenwereld

In de land- en tuinbouw heeft de ontwikkeling van (moleculaire) detectiemethoden van plantenpathogenen de laatste jaren een hoge vlucht genomen. Inmiddels worden deze methoden al grootschalig toegepast in de praktijk. Werd in het begin alleen conventionele polymerase chain reaction (PCR) ingezet voor moleculaire detectie, momenteel vindt ook real-time PCR meer en meer ingang. Binnen het FES-programma ‘Versterking infrastructuur plantgezondheid’ zijn binnen het werkpakket ‘Identificatie- en Detectiemethoden’ vele projecten uitgevoerd om de ‘daders’ van aantastingen te kunnen identificeren. De focus was hierbij gericht op quarantaineorganismen