PubMed · 8251393
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
R E Gale, J S Wainscoat. 1993. Clonal analysis using X-linked DNA polymorphisms.. https://doi.org/10.1111/j.1365-2141.1993.tb08637.x
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.
Explore the source record for details and available documents.
DNA and RNA probes are important analytical reagents in molecular biology and in the detection of infectious and genetic diseases. However, the present polynucleotide probe technology is complex and labor-intensive. We have been investigating the possibility of using fluorescent-labeled DNA probes to develop assays which do not require the separation of free from hybridized probe (homogeneous assays). Such assays are possible if the fluorescence efficiency or fluorescence anisotropy of the fluorescent label changes upon hybridization of probe with target DNA. In this article we examine pyrene as a fluorescent label for DNA or RNA probes. Experiments were performed using a model system in which poly(C) and poly(I) are respectively the probe and target sequences. A small fraction of the nucleotide bases of poly(C) was randomly labeled with pyrene using the bisulfite-catalyzed diamine reaction. The results show that the uncorrected emission spectrum of pyrene-poly(C) decreases by a factor of 4 and shifts toward longer wavelengths upon hybridization with poly(I) at saturating concentrations. The average lifetime changes from 10.78 to 4 ns. These fluorescence changes occur in a wide range of chemical environments, including the high salt concentrations normally used to increase the velocity of the hybridization reaction in clinical assays. The pyrene label can thus be used to readily detect the amount of poly(I) in an unknown sample without having to separate free and bound labeled probe. To unravel the mechanism responsible for the observed changes in fluorescence intensity upon hybridization, we have performed polarized fluorescence intensity measurements and analyzed the results by approximate steady-state expressions that allow evaluation of the relative contributions of changes in lifetimes (fluorescence efficiency) and rotational motions to the changes in fluorescence intensity. The results indicate that the latter changes are due chiefly to changes in lifetime or fluorescence efficiency and that these changes seem to be due to the movement of the pyrene label to a more hydrophilic environment upon hybridization.