生物信息学技术

Forced intercalation probes (FIT-probes) are peptide nucleic acid-based probes in which the thiazole orange dye replaces a canonical nucleobase. FIT-probes are used in homogenous DNA detection. The analysis is based on sequence-specific binding of the FIT-probe with DNA. Binding of t ...

Fluorescent nucleic acid detection in polymerase chain reaction (PCR) generally uses oligonucleotide probes labeled with covalently attached dyes. However, unlabeled oligonucleotides in the presence of saturating DNA dyes can also serve as hybridization probes. The DNA dye, L ...

Real-time monitoring of DNA—protein interactions involving molecular beacon (MB) and molecular beacon aptamer (MBA) was discussed in this chapter. MBs are single-stranded oligonucleotide probes with a hairpin structure. MBs have been designed for oligonucleotide recognit ...

We report a simple homogeneous fluorescence assay for quantification of DNA polymerase function in high throughput. The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon extension of the template strand. A resulting distance alterati ...

Methods for the detection of biologically relevant interactions by highly precise catalytic control elements based on hairpin ribozymes, and their subsequent analysis are described. These include ribozyme design, catalytic performance in real time as a function of fluorescen ...

The characterization of molecular interactions is a central task in modern life sciences. Applications such as drug screening in pharmaceutics or the elucidation of biomolecular interactions in molecular biology rely on efficient methods to search for interacting partners. Her ...

Industrial manufacturing of cell culture-derived viruses or virus-like particles for gene therapy or vaccine production are complex multistep processes. In addition to the bioreactor, such processes require a multitude of downstream unit operations for product separation, c ...

The purification of the product, the so-called downstream process (DSP), tends to be one of the most costly aspects of modern bioprocessing, especially in the case of proteins. In such cases, chromatography is still the major tool on all levels of the DSP from the first capture to the final polishing step. ...

The use of baculoviruses has become a standard approach in many labs for recombinant protein production. In addition to giving a broad and practical overview of the technology, this chapter focuses in particular on two recent developments in the field and how these can be efficiently exploited ...

The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as w ...

For the production of biopharmaceuticals a seed train is required to generate an adequate number of cells for inoculation of the production bioreactor. This seed train is time- and cost-intensive but offers potential for optimization. A method and a protocol are described for the seed train ma ...

This protocol regards a microscopic application and software for image-guided monitoring of mammalian cells which grow in suspension cultures. It has been developed in order to establish an automated microscopic application for in situ and at-line cell monitoring in bioreactors (A ...

Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize ...

Microfluidic perfusion culture is a novel technique to culture animal cells in a small-scale microchamber with medium perfusion. Polydimethylsiloxane (PDMS) is the most popular material to fabricate a microfluidic perfusion culture chip. Photolithography and replica moldi ...

1H-Nuclear magnetic resonance (1H-NMR) spectroscopy is a powerful technique to analyze the composition of complex mixtures based on the particular proton fingerprint of each molecule. Here we describe a protocol for exometabolome analysis of mammalian cells using this technique, ...

Metabolomics has become an important tool for measuring pools of small molecules in mammalian cell cultures expressing therapeutic proteins. NMR spectroscopy has played an important role, largely because it requires minimal sample preparation, does not require chromatograp ...

Sampling and quenching methods for intracellular metabolite analysis in mammalian cells in adherent and suspension culture are described. Quenching of adherent cells is achieved by application of hot air after removal of the supernatant by suction. For suspension cultures, the add ...

The accurate determination of cell growth and viability is pivotal to monitoring a bioprocess. Direct methods to determine the cell growth and/or viability in a bioprocess include microscopic counting, electronic particle counting, image analysis, in situ biomass monitoring, and d ...

The majority of therapeutic proteins are expressed in mammalian cells, predominantly in Chinese Hamster Ovary cells. While cell culture media and feed supplements are crucial to protein productivity, medium optimization can be labor intensive and time-consuming. In this chapter, we ...

Fed-batch processes are a current preference for the production of recombinant proteins in mammalian cells. The use of nutrient feeding prevents the depletion of important medium components and results in improved culture longevity and high cell and product yields. To take maximum adv ...