A method for nucleic acid hybridization to isolated chromosomes in suspension

A procedure was developed to provide differential fluorescent staining of metaphase chromosomes in suspension following nucleic acid hybridization. For this purpose metaphase chromosomes were isolated from a Chinese hamster x human hybrid cell line. After hybridization with biotinylated human genomic DNA, the human chromosomes were visualized by indirect immunofluorescence using antibodies against biotin and fluoresceine-isothiocyanate-(FITC)-labeled second antibodies. This resulted in green fluorescent human chromosomes. In contrast, Chinese hamster chromosomes revealed red fluorescent staining only when counterstained with propidium iodide. Notably, interspecies chromosomal rearrangements could be easily detected. After hybridization and fluorescent staining, chromosomes still showed a well-preserved morphology under the light microscope. We suggest that this procedure may have a useful application in flow cytometry and sorting

On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode

Many pharmaceuticals are proteins or their development is based on proteins. Cell-free protein synthesis (CFPS) is an innovative alternative to conventional cell based systems which enables the production of proteins with complex and even new characteristics. However, the short lifetime, low protein production and expensive reagent costs are still limitations of CFPS. Novel automated microfluidic systems might allow continuous, controllable and resource conserving CFPS. The presented microfluidic TRITT platform (TRITT for Transcription - RNA Immobilization & Transfer - Translation) addresses the individual biochemical requirements of the transcription and the translation step of CFPS in separate compartments, and combines the reaction steps by quasi-continuous transfer of RNA templates to enable automated CFPS. In detail, specific RNA templates with 5' and 3' hairpin structures for stabilization against nucleases were immobilized during in vitro transcription by newly designed and optimized hybridization oligonucleotides coupled to magnetizable particles. Transcription compatibility and reusability for immobilization of these functionalized particles was successfully proven. mRNA transfer was realized on-chip by magnetic actuated particle transfer, RNA elution and fluid flow to the in vitro translation compartment. The applicability of the microfluidic TRITT platform for the production of the cytotoxic protein Pierisin with simultaneous incorporation of a non-canonical amino acid for fluorescence labeling was demonstrated. The new reaction mode (TRITT mode) is a modified linked mode that fulfills the precondition for an automated modular reactor system. By continual transfer of new mRNA, the novel procedure overcomes problems caused by nuclease digestion and hydrolysis of mRNA during TL in standard CFPS reactions.BMBF, 0312039, Nachwuchsgruppe- Biotechnologie 2020+: Chip-basierter Automat für die zellfreie ProteinsyntheseBMBF, 0315942, Zellfreie Bioproduktion - Etablierung einer Bioproduktionsanlage für die zellfreie Proteinsynthese mit integrierter Energieversorgung - Biomoleküle vom Ban

Immobilization of different biomolecules by atomic force microscopy

<p>Abstract</p> <p>Background</p> <p>Micrometer resolution placement and immobilization of probe molecules is an important step in the preparation of biochips and a wide range of lab-on-chip systems. Most known methods for such a deposition of several different substances are costly and only suitable for a limited number of probes. In this article we present a flexible procedure for simultaneous spatially controlled immobilization of functional biomolecules by molecular ink lithography.</p> <p>Results</p> <p>For the bottom-up fabrication of surface bound nanostructures a universal method is presented that allows the immobilization of different types of biomolecules with micrometer resolution. A supporting surface is biotinylated and streptavidin molecules are deposited with an AFM (atomic force microscope) tip at distinct positions. Subsequent incubation with a biotinylated molecule species leads to binding only at these positions. After washing streptavidin is deposited a second time with the same AFM tip and then a second biotinylated molecule species is coupled by incubation. This procedure can be repeated several times. Here we show how to immobilize different types of biomolecules in an arbitrary arrangement whereas most common methods can deposit only one type of molecules. The presented method works on transparent as well as on opaque substrates. The spatial resolution is better than 400 nm and is limited only by the AFM's positional accuracy after repeated z-cycles since all steps are performed in situ without moving the supporting surface. The principle is demonstrated by hybridization to different immobilized DNA oligomers and was validated by fluorescence microscopy.</p> <p>Conclusions</p> <p>The immobilization of different types of biomolecules in high-density microarrays is a challenging task for biotechnology. The method presented here not only allows for the deposition of DNA at submicrometer resolution but also for proteins and other molecules of biological relevance that can be coupled to biotin.</p

Self-similarity and non-Markovian behavior in traded stock volumes

The volume traded daily for 17 stocks is followed over a period of about half a century. We look at the volume of stocks traded in a certain time interval (day, week, month) and analyze how long that traded volume keeps monotonically increasing or decreasing. On all three times scales we find that the sequence of traded volumes behaves neither like a sequence of independent and identically distributed variables, nor like a Markov sequence. A compressed exponential survival function with the same parameters at all timescales is firmly established. A day with an increase (decrease) of traded volume is most likely followed by a day with a decrease (increase) of traded volume. We show how the apparent self-similarity results because the small day-to-day anticorrelation carries over when larger time intervals are considered. The observed small anticorrelation can be explained as a consequence of market forces and trader reactions

Tear proteome analysis in ocular surface diseases using label-free LC-MS/MS and multiplexedmicroarray biomarker validation

We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers employing customized antibody microarray assays. Comparative analysis of tear protein profiles revealed differences in the expression levels of 26 proteins, including protein S100A6, annexin A1, cystatin-S, thioredoxin, phospholipase A2, antileukoproteinase, and lactoperoxidase. Antibody microarray validation of CST4, S100A6, and MMP9 confirmed the accuracy of previously reported ELISA assays, with an area under ROC curve (AUC) of 87.5%. Clinical endpoint analysis showed a good correlation between biomarker concentrations and clinical parameters. In conclusion, different sets of proteins differentiate between the groups. Apolipoprotein D, S100A6, S100A8, and ceruloplasmin discriminate best between the DE and CT groups. The differences between antileukoproteinase, phospholipase A2, and lactoperoxidase levels allow the distinction between MGD and DE, and the changes in the levels of annexin A1, clusterin, and alpha-1-acid glycoprotein 1, between MGD and CT groups. The functional network analysis revealed the main biological processes that should be examined to identify new candidate biomarkers and therapeutic targets

Construction of an artificial cell membrane anchor using DARC as a fitting for artificial extracellular functionalities of eukaryotic cells

The need to functionalize cell membranes in a directed way for specific applications as single cell arrays or to force close cell-to-cell contact for artificial intercellular interaction and/or induction concerning stem cell manipulation or in general to have a tool for membrane and cell surface-associated processes, we envisaged a neutral inactive membrane anchor for extracellular entities to facillitate the above mentioned functionalities

Label-free electrical quantification of the dielectrophoretic response of DNA

A purely electrical sensing scheme is presented that determines the concentration of macromolecules in solution by measuring the capacitance between planar microelectrodes. Concentrations of DNA in the ng/mL range have been used in samples of 1 microL volume. The method has been applied to the characterisation of the dielectrophoretic response of DNA without the need for any chemical modifications. The influence of electrical parameters like duty cycle, voltage and frequency has been investigated. The results are in good agreement with data from dielectrophoretic studies on fluorescently labelled DNA. Extension of the method down to the single molecule level appears feasible.Comment: 12 pages, 7 figure

DNA-nanostructure-assembly by sequential spotting

<p>Abstract</p> <p>Background</p> <p>The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions.</p> <p>Results</p> <p>For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM) has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid) to incorporate advanced structures.</p> <p>Conclusions</p> <p>The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element.</p