78 research outputs found
Integrated modular microfluidic system for forensic Alu DNA typing
Driven by the numerous applications of genome-related research, fully integrated microfluidic systems have been developed that have advanced the capabilities of molecular and, in particular, genetic analyses. A brief overview on integrated microfluidic systems for DNA analysis is given in Chapter 1 followed by a report on micro-capillary electrophoresis (µCE) of Alu elements with laser-induced fluorescence (LIF) detection, in which the monomorphic Alu insertions on the X and Y chromosomes were utilized to detect male DNA in large female DNA background (Y: X = 1:19) without cell sorting prior to the determination. The polymorphic Alu loci with known restricted geographical distribution were used for ethnicity determination. A valveless integrated microsystem that consists of three modules is discussed as well: (1) A solid-phase extraction (SPE) module microfabricated on polycarbonate, for DNA extraction from whole cell lysates (extraction bed capacity ~209 ±35.6 ng/cm² of total DNA). (2) A continuous-flow polymerase chain reaction (CFPCR) module fabricated in polycarbonate (Tg ~150 ºC) in which selected gene fragments were amplified using biotin and fluorescently-labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones. (3) µCE module fabricated in poly(methylmethacrylate), which utilized a bioaffinity selection and purification bed (2.9-µL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to µCE. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed where they were extracted onto the surface of the poly(methylmethacrylate) micropillars (50-µm width; 100-µm height; total surface area of ~117 mm²). This SPE process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (Kd =10-15M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single stranded DNA released for size-based separations and LIF detection. The multiplexed SPE-CFPCR-µCE yielded detectable fluorescence signal (S/N≥3; LOD ~75 cells) for Alu DNA amplicons for gender and ethnicity determinations with a separation efficiency of ~1.5 x105 plates/m. Compared to traditional cross-T injection procedures typically used for µCE, the affinity preconcentration and injection procedure generated signal enhancements of 17-40 fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation
Characterization of the tumor supressor activity of the FHIT gene in association with application of innovative detection technologies
In early 1996, the Fragile Histidine Triad (FHIT) gene was cloned and shown to straddle the most active of the fragile human chromosome sites at chromosome band 3pl4.2. The exceptionally large FHIT locus encompasses a hereditary renal carcinoma-associated chromosome translocation breakpoint. The size of the FHIT gene is about 1 megabase of genomic DNA, encoding a 1.1 kb niRNA message and a 16.8 kDa protein with diadenosine triphosphate hydrolase activity.
Early studies of a number of important human tumors such as breast, gastric, renal, and lung carcinoma and pancreatic adenocarcinoma have revealed that FHIT RNA expression was frequently altered and these alterations correlated with deletions in the FHIT gene, suggesting a role for this gene in development of cancer. There has also been a correlation between complete absence of Fhit protein and the early clinical stages of cancer. Such observations implicated FHIT as a putative tumor suppressor gene. Nonetheless, several lines of evidence have called into question the role of FHIT as a classical tumor suppressor gene, and raised the question of whether its apparent involvement simply reflects its location within an unstable region of the genome. Observation of biallelic deletions rather than mutations of the FHIT gene in cancers prompted a number of investigators to reject FHIT as a suppressor gene. In addition, consistent effects of exogenous FHIT on growth in cultures had not been observed. Additionally, experiments transfecting wild-type (wi) FHIT into tumor cell lines with FHIT abnormalities have produced conflicting results regarding suppression of tumorigenesis in vivo. The primary objective of this project was to investigate whether the FHIT gene was indeed a tumor suppressor gene. The goal was to establish a representation of the underlying molecular and cellular mechanisms of action of FHIT gene in suppression of tumorigenesis. The results demonstrate that the FHIT gene is indeed a tumor suppressor gene and that Fhit expression plays a possible role in induction of apoptosis. Our data indicates that Fhit protein alters the mitochondrial flux and efflux of molecules causing alterations in the transmembrane potential in the presence of apoptotic stimuli. We observed that induction of apoptosis in cells expressing the Fhit protein also involved the release of mitochondrial cytochrome c from the mitochondria and its subsequent translocation into the cytoplasmic compartment.
This investigation was also aimed at developing and applying novel spectroscopic and biosensing techniques and protocols designed to provide alternative methods for gene and protein identification, and defining gene function at the cellular and molecular levels for applications in biological research and clinical diagnosis laboratories. For the design of these systems, we took advantage of optical spectroscopy techniques including fluorescence, and synchronous luminescence spectroscopy, the biochip technology, and various microscopy methods. These modern technologies, developed at ORNL, have the potential to be selective, as well as sensitive, in providing information to understand how gene expression impacts a specific biological system
Lab-on-a-Chip Fabrication and Application
The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application
Development of micro total analysis system for detection of water pathogens
Master'sMASTER OF ENGINEERIN
Multiplexed profiling of extracellular vesicles for biomarker development
Extracellular vesicles (EVs) are cell-derived membranous particles that play a crucial role in molecular trafficking, intercellular transport and the egress of unwanted proteins. They have been implicated in many diseases including cancer and neurodegeneration. EVs are detected in all bodily fluids, and their protein and nucleic acid content offers a means of assessing the status of the cells from which they originated. As such, they provide opportunities in biomarker discovery for diagnosis, prognosis or the stratification of diseases as well as an objective monitoring of therapies. The simultaneous assaying of multiple EV-derived markers will be required for an impactful practical application, and multiplexing platforms have evolved with the potential to achieve this. Herein, we provide a comprehensive overview of the currently available multiplexing platforms for EV analysis, with a primary focus on miniaturized and integrated devices that offer potential step changes in analytical power, throughput and consistency
Microfluidic technologies for genomic interrogation of mycobacterium tuberculosis clinical isolates using the polymerase chain reaction (PCR) and high resolution melting analysis (HRMA).
Master of Medical Science in Medical Microbiology. University of KwaZulu-Natal, Medical School 2015.Background: A number of Mycobacterium tuberculosis (Mtb) genes have been shown to be under positive selection pressure in the presence of anti-TB therapy. This results in the selection of drug resistant phenotypes associated with genetic changes—which can be point mutations, deletions and/or insertions. Some mutations from multiple genes have been documented to be associated with reduced susceptibility to anti-TB drugs such as rifampicin, ethambutol, carpreomycin and fluoroquinolones. The list is continuously updated as new mutations are discovered and validated. In principle therefore, there is an urgent need to design robust molecular diagnostics and more efficacious therapeutic strategies that are able to indicate diverse genetic mechanisms behind drug resistance in individual isolates
Materials and Methods: We used the LightForge system we developed at K-RITH. This LightForge system is a fluorescence detection based, highly scalable microfluidic platform. It interrogates Mycobacterium tuberculosis strains using Real-Time PCR and High Resolution Melt Analysis (HRMA) on a chip.
Results and Discussion: We have used this LightForge system to identify clinical Mtb strains resistant to rifampicin—a frontline drug used to treat tuberculosis, relative to a susceptible strain H37RV, based on mutations in the rpoB gene. This system has the potential to contribute towards a low-cost solution to diagnosis of multidrug resistant tuberculosis—a current critical global healthcare challenge. The interrogation of clinical Mtb isolates—including R35, KZN 605 and Tkk 01-062—using the LightForge system has detected mutations linked to rifampicin resistance including single nucleotide polymorphisms (SNPs) in a congruous manner with commercial systems.
Conclusions: In preparation for diagnosis of clinical samples, this LightForge approach is now being expanded to incorporate detection of genetic markers linked with resistance to other TB drugs that include fluoroquinolones and isoniazid based on mutations in gyrA, katG and Mab-inhA regions of the Mtb genome. The scalability of LightForge can also be harnessed to conduct digital PCR (dPCR), a critical tool for detecting genetic heterogeneity in Mtb
Von Plattformen zu miRNA-Biomarkern : Methoden zur miRNA-Molekulardiagnostik
An obvious way to improve human healthcare is to develop new and more effective drugs.
Another opportunity is however to develop solutions that allow to utilize the available
drugs better. This includes more accurate and early diagnosis of pathologies, improved
therapy selection as well as digital and patient centric solutions in healthcare systems.
Especially in molecular diagnostics new biomarkers have been developed and partially
shown promising results in terms of improving patient care. In this work I describe the
development of respective platform techniques, biomarkers and computational solutions
during my PhD thesis.
First, I briefly introduce the concept of a flexible microarray platform and assays, such as
the MPEA assay, tailored for the fast and efficient quantification of miRNA signatures.
Then, I describe how we made use of respective platforms along with computational
solutions to improve the understanding of physiological and pathophysiological
processes. Further, I present results on my efforts to develop new molecular diagnostic
biomarkers based on circulating miRNAs. Here, my special focus was in cancer (most
importantly lung cancer) and diseases affecting the Central Nervous System (most
importantly Multiple Sclerosis, Alzheimer’s Disease and Parkinson’s Disease). Together
with the supervisors of my thesis I was among the first researchers worldwide to
recognize that small non-coding RNAs (most importantly microRNAs) measured from
body fluids have a great potential as biomarkers. An obvious advantage to messenger
RNAs is the small length of the molecules of only 17-22 nucleotides. This makes
microRNAs stable in vivo but also in vitro.
Finally, I will mention recent developments in patient care. The current trend is clearly
the digitalization of central parts of healthcare. This affects all stakeholders in the
healthcare system, most importantly medical doctors and patients. Especially patient
empowerment and self-containment of medical data is becoming more important. Again,
Multiple Sclerosis is used as an example. But also for physicians, computational tools have
to be implemented to support them in making treatment decisions from highly complex
data. In sum, my thesis describes the road from developing a molecular diagnostic
platform over the research on biomarkers for detecting disease in time towards holistic
computational solutions to improve patient care.Es ist offensichtlich, dass man Krankheiten besser behandeln kann, wenn man neue und
effektivere Medikamente und Therapien entwickelt. Eine andere Möglichkeit ist es,
Lösungen zu entwickeln, die es erlauben, vorhandene Medikamente besser einzusetzen.
Das schließt die frühzeitige Diagnose von Erkrankungen, eine verbesserte Wahl der
richtigen Therapie und die Entwicklung von patienten-zentrischen digitalisierten
Lösungen mit ein. Insbesondere in der Molekulardiagnostik wurden neue
vielversprechende Biomarker entwickelt. In dieser Arbeit führe ich meine Beiträge zur
Entwicklung von Plattform Technologien zum Messen von Biomarkern aus, erläutere die
Erforschung von Biomarkern selbst und beschreibe die Anwendung der dazugehörigen,
computergestützten Methoden.
Beginnen möchte ich mit einer Beschreibung der Entwicklung einer flexiblen Mikroarray
Plattform und Assays, wie zum Beispiel des MPEA Assays, die maßgeschneidert für die
schnelle und effiziente Quantifizierung von miRNA Biomarkern sind. Dann gehe ich darauf
ein, wie wir Plattformen, Assays und computergestützte Lösungen eingesetzt haben, um
physiologische und pathologische Prozesse besser zu verstehen. Außerdem präsentiere
ich Resultate meiner Bemühung, neue molekulardiagnostische Biomarker basierend auf
zirkulierenden miRNA Mustern zu entwickeln. Hierbei habe ich mich auf Krebs
(vornehmlich Lungentumore) und Erkrankungen, die das Zentrale Nervensystem
betreffen (Multiple Sklerose und die Alzheimer Erkrankung), konzentriert. Gemeinsam
mit meinen Betreuern war ich unter den ersten Forschern weltweit, die das große
Potenzial kleiner nicht-kodierender RNAs (am wichtigsten dabei microRNAs), die aus
Blut gemessen werden können, erkannt haben. Ein offensichtlicher Vorteil gegenüber
mRNA Biomarkern ist die kurze Länge von nur 17-22 Nukleotiden. Diese macht miRNAs
sowohl in-vivo als auch in-vitro stabil.
Letztlich gehe ich in meiner Arbeit auf momentane Entwicklungen in der
Patientenversorgung ein. Ein klarer Trend ist die Digitalisierung zentraler Teile der
Gesundheitsversorgung. Das betrifft alle Personen im Gesundheitswesen, allen voran
Mediziner und Patienten. Selbstbestimmung des Patienten wird besonders wichtig
werden. Hier dient mir wieder Multiple Sklerose als ein Beispiel. Auch für Ärzte müssen,
angesichts der immer komplexeren Daten, computergestützte Lösungen entwickelt
werden, die ihnen helfen, die richtige Therapieentscheidung zu treffen.
Zusammenfassend halte ich fest, dass meine Arbeit den Weg von der Entwicklung einer
molekulardiagnostischen Plattform über die Entwicklung von Biomarkern zur
Frühdiagnose von Erkrankungen bis hin zu ganzheitlichen computergestützten
Lösungen, die die Patientenversorgung verbessern, beschreibt
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