PubMed · 9664470
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S Viville, R Mantovani. 1998. S1 mapping using single-stranded DNA probes.. https://doi.org/10.1385/0-89603-494-1%3A195
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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.
The application of resonance light scattering (RLS) particles for high-sensitivity detection of DNA hybridization on cDNA microarrays is demonstrated. Arrays composed of approximately 2000 human genes ("targets") were hybridized with colabeled (Cy3 and biotin) human lung cDNA probes at concentrations ranging from 8.3 ng/microL to 16.7 pg/microL. After hybridization, the arrays were imaged using a fluorescence scanner. The arrays were then treated with 80-nm-diameter gold RLS Particles coated with anti-biotin antibodies and imaged in a white light, CCD-based imaging system. At low probe concentrations, significantly more genes were detected by RLS compared to labeling by Cy3. For example, for hybridizations with a probe concentration of 83.3 pg/microL, approximately 1150 positive genes were detected using RLS compared to approximately 110 positive genes detected with Cy3. In a differential gene expression experiment using human lung and leukemia RNA samples, similar differential expression profiles were obtained for labeling by RLS and fluorescence technologies. The use of RLS Particles is particularly attractive for detection and identification of low-abundance mRNAs and for those applications in which the amount of sample is limited.
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