PubMed · 9193720
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D S Millican, I M Bird. 1997-06-15. A general method for single-stranded DNA probe generation.. https://doi.org/10.1006/abio.1997.2163
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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.
Fusarium head blight (FHB) of wheat is a crippling disease that causes severe economic losses in many of the wheat-growing regions of the world. Temporal patterns of fungus development and transcript accumulation of defense response genes were studied in Fusarium graminearum-inoculated wheat spikes within the first 48 to 76 h after inoculation (hai). Microscopy of inoculated glumes revealed that the fungus appeared to penetrate through stomata, exhibited subcuticular growth along stomatal rows, colonized glume parenchyma cells, and sporulated within 48 to 76 hai. No major differences in the timing of these events were found between Sumai 3 (resistant) and Wheaton (susceptible) genotypes. In complementary experiments, RNA was extracted from spikes at several time intervals up to 48 hai and temporal expression patterns were determined for defense response genes encoding peroxidase, PR-1, PR-2 (beta-1,3-glucanase), PR-3 (chitinase), PR-4, and PR-5 (thaumatin-like protein). In both genotypes, transcripts for the six defense response genes accumulated as early as 6 to 12 hai during F. graminearum infection and peaked at 36 to 48 hai. Greater and earlier PR-4 and PR-5 transcript accumulation was observed in Sumai 3, compared with Wheaton. Our results show that the timing of defense response gene induction is correlated with F. graminearum infection.
Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) retrieved two clusters of sequences resembling sulfate-reducing bacteria within the family Desulfovibrionaceae. In situ hybridization showed that, similar to sulfate-reducing bacteria of the family Desulfobacteriaceae, bacteria of one cluster with similarity values to the closest cultured relatives of between 92.6 and 93.1% resembled free cells or cells loosely attached to other cells or debris. Bacteria of the second cluster closely related to Desulfocapsa thiozymogenes DSM7269 with similarity values between 97. 9 and 98.4% were generally associated with aggregates of different small-celled phototrophic sulfur bacteria, suggesting a potential interaction between the two groups of bacteria.