ABSTRACT
Microarray technology provides new analytical devices that allow the parallel and simultaneous detection of several thousands of probes within one sample. Microarrays, sometimes called DNA chips, are widely used in gene-expression analysis, genotyping of individuals, analysis of point mutations and single nucleotide polymorphisms (SNP) as well as other genomic or transcriptomic variations. In this chapter we give a survey of common microarray manufacturing, the selection of support material, immobilisation and hybridisation and the detection with labelled complementary strands. However, DNA arrays may also serve as the basis for more complex analysis based on the action of enzymes on the immobilized templates. This property gives DNA microarrays the potential for being the template for whole PCR and transcription experiments with high parallelism, as will be discussed in the last section of this chapter.
Subject(s)
Oligonucleotide Array Sequence Analysis/methods , DNA Mutational Analysis/instrumentation , DNA Mutational Analysis/methods , Oligonucleotide Array Sequence Analysis/instrumentation , Point Mutation , Polymerase Chain Reaction/instrumentation , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Transcription, GeneticABSTRACT
Growth factors are an important tool in tissue engineering. Bone morphogenetic protein-2 and transforming growth factor-beta(1) (TGF-beta(1)) are used to provide bioactivity to surgical implants and tissue substitute materials. Mostly growth factors are used in soluble or adsorbed form. However, simple adsorption of proteins to surfaces is always accompanied by reduced stability and undefined pharmacokinetics. This study aims to prove that TGF-beta(1) can be covalently immobilized to functionalized surfaces, maintaining its ability to induce myofibroblastic differentiation of normal human dermal fibroblasts. In vivo, fibroblasts differentiate to myofibroblasts (MFs) during soft tissue healing by the action of TGF-beta(1). As surfaces for our experiments, we used slides bearing aldehyde, epoxy, or amino groups. For our in vitro cell culture experiments, we used the expression of alpha-smooth muscle actin as a marker for MFs after immunochemical staining. Using the aldehyde and the epoxy slides, we were able to demonstrate the activity of immobilized TGF-beta(1) through a significant increase in MF differentiation rate. A simple immunological test was established to detect TGF-beta(1) on the surfaces. This technology enables the creation of molecular "landscapes" consisting of several factors arranged in a distinct spatial pattern and immobilized on appropriate surfaces.
Subject(s)
Cell Differentiation/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Transforming Growth Factor beta1/pharmacology , Actins/metabolism , Biomarkers/metabolism , Cell Differentiation/physiology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Fibroblasts/physiology , Fluorescent Dyes/metabolism , Humans , Hydrogen-Ion Concentration , Immunohistochemistry , Indoles/metabolism , Muscle, Smooth/chemistry , Propylamines , Silanes/chemistry , Skin/cytology , Substrate Specificity , Surface Properties , Tissue Engineering/methods , Transforming Growth Factor beta1/analysis , Transforming Growth Factor beta1/chemistryABSTRACT
Our experiments describe an alternative method of dsDNA recognition using zinc finger (ZF) molecules which bind DNA specifically and with high affinity. Our aim was to develop zinc finger probes which are able to bind to dsDNA molecules at predetermined sites. In our basic approach we used pairs of complementary oligonucleotides to form dsDNAs, containing one of the three SP1-transcription factor motifs as a zinc finger recognition site. Two zinc finger probes of the SP1 motif were chemically synthesized and modified with a Dy-633 fluorophore. The SP1 peptides were folded into functional zinc fingers using zinc chloride. The addressable dsDNAs were immobilized on optical fibres, and the kinetics and binding rates of the artificial zinc finger probes were measured by a fluorescence detecting device (photomultiplying tube). The two zinc fingers and their corresponding DNA recognition sites served as specific probes and controls for the matching site and vice versa. Our experiments showed that a variety of dsDNA-binding probes may be created by modification of the amino acid sequence of natural zinc finger proteins. Our findings offer an alternative approach of addressing dsDNA molecules, for example for use in a nanoarray device.
