BackgroundIntroduction of microbeads into flow‐cytometry has created a new scenario, making quantitative measurement of molecules dispersed in a homogeneous phase, with an extremely wide realm of already realized and potential applications possible. Development of this field has lead to specialized instrumentation and microbead arrays, dedicated to certain applications.MethodsFormaldehyde‐fixed yeast and bacterial cells were conjugated with avidin and applied as microbeads, to establish a simple, convenient, flexible, and inexpensive flow‐cytometric platform for various immunological and biochemical assays.ResultsWe have tested these “biological microbeads” for the simultaneous titration of human α‐fetoprotein (AFP) and human Chorionic Gonadotropin (βhCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers.ConclusionsThe use of biological microbeads for various immunological and biochemical assays has been demonstrated. The flow‐cytometric methods proved to be at least as sensitive as the standard biochemical or immunological tests. For proteinase K activity measurements, a single enzyme molecule in the sample could be detected. The sensitivity, versatility, and low cost of the assays may advance flow‐cytometry to become a central methodological platform in most laboratories. The biological microbeads offer virtually unlimited possibilities for fluorescent labeling (addressing), conjugation of ligand binding molecules, and they are easy to handle and perform well in a multiplex format. © 2005 International Society for Analytical Cytology
We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin-and dye labeled reaction products are immobilized on microbeads and the homo-and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero-from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single-strand lesions formed at hypersensitive sites in response to apoptotic stimuli. ' 2008 International Society for Analytical Cytology Key terms DNA; deletion; insertion; mismatch; nick-translation; flow cytometry; S1 nuclease; chemical cleavage; MLL ENCOURAGED by favorable earlier experience (1-4), we explore the possibilities offered by microbead based flow cytometric technology to perform various forms of molecular biological analysis. Here we demonstrate strategies to detect the frequent double-strand (ds) genetic lesions, insertions and deletions, as well as certain singlestrand (ss) lesions, using a flow cytometric platform. Deletions and insertions can be readily recognized as a result of the changes in the length of PCR products encompassing the particular region. A flow cytometric read-out of such data may offer high throughput (HTP) potential perspective and may also prove useful when small differences are to be recognized; in addition, this approach may be more cost-effective than sequencing in certain cases. Using a model system, the breakpoint-cluster region (bcr) of the mixed lineage leukemia (MLL) gene, we demonstrate that heteroduplices formed after denaturation and reannealing of PCR products of different size are preferentially cleaved by the ss specific nuclease S1, and also by chemical reactions specific for unpaired nucleotides: on the other hand, restriction enzymes are unable to cleave their recognition motives in the overhang area. These reactions lead to significantly altered average fluorescence of the streptavidinated microbeads anchoring the hybrids through their biotinylated ends.
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