RESUMO
The protocols for in situ hybridization (ISH) techniques can vary considerably; however, they usually include denaturation and hybridization steps. Denaturing compounds are used to reduce denaturation and hybridization temperature, which keeps the proper morphology of the preparation. Formamide is the most commonly used reagent in in situ hybridization to lower the melting temperature. The substitution of toxic formamide for a non-toxic ethylene carbonate at 20 % and 50% concentration in the hybridization mixture helped obtain a high quality in situ hybridization result with two sequences characteristic for rye, JNK, and Bilby. The results after hybridization, with a duration of 90 min and 16 h, were identical when formamide or ethylene carbonate were used in the mixture. In addition, the toxic formamide was eliminated from the post-hybridization steps and specific hybridization signals for both probes were still obtained.
Assuntos
Hibridização In Situ/métodos , Hibridização In Situ/normas , Indicadores e Reagentes/administração & dosagem , Secale/efeitos dos fármacos , Secale/genéticaRESUMO
The protocols for in situ hybridization (ISH) techniques can vary considerably; however, they usually include denaturation and hybridization steps. Denaturing compounds are used to reduce denaturation and hybridization temperature, which keeps the proper morphology of the preparation. Formamide is the most commonly used reagent in in situ hybridization to lower the melting temperature. The substitution of toxic formamide for a non-toxic ethylene carbonate at 20 % and 50% concentration in the hybridization mixture helped obtain a high quality in situ hybridization result with two sequences characteristic for rye, JNK, and Bilby. The results after hybridization, with a duration of 90 min and 16 h, were identical when formamide or ethylene carbonate were used in the mixture. In addition, the toxic formamide was eliminated from the post-hybridization steps and specific hybridization signals for both probes were still obtained.(AU)
Assuntos
Hibridização In Situ/métodos , Hibridização In Situ/normas , Secale/efeitos dos fármacos , Indicadores e Reagentes/administração & dosagem , Secale/genéticaRESUMO
In plants, partial DNA sequences of chloroplasts have been widely used in evolutionary studies. However, the Cactaceae family (1500-1800 species) lacks molecular markers that allow a phylogenetic resolution between species and genera. In order to identify sequences with high variation levels, we compared previously reported complete chloroplast genomes of seven species of Mammillaria. We identified repeated sequences (RSs) and two types of DNA variation: short sequence repeats (SSRs) and divergent homologous loci. The species with the highest number of RSs was M. solisioides (256), whereas M. pectinifera contained the highest amount of SSRs (84). In contrast, M. zephyranthoides contained the lowest number (35) of both RSs and SSRs. In addition, five of the SSRs were found in the seven species, but only three of them showed variation. A total of 180 homologous loci were identified among the seven species. Out of these, 20 loci showed a molecular variation of 5% to 31%, and 12 had a length within the range of 150 to 1000 bp. We conclude that the high levels of variation at the reported loci represent valuable knowledge that may help to resolve phylogenetic relationships and that may potentially be convenient as molecular markers for population genetics and phylogeographic studies.