Heteroduplex analysis using flow cytometric microbead assays to detect deletions, insertions, and single-strand lesions.

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.

Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR.

BACKGROUND: Introduction 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. METHODS: Formaldehyde-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. RESULTS: We have tested these "biological microbeads" for the simultaneous titration of human alpha-fetoprotein (AFP) and human Chorionic Gonadotropin (betahCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers. CONCLUSIONS: The 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.