RESUMO

Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/ß-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications.

Assuntos

DNA Fúngico/isolamento & purificação , Fungos/genética , Genômica/métodos , Misturas Complexas/isolamento & purificação , Reação em Cadeia da Polimerase

RESUMO

Here, it is presented a rapid and efficient method to obtain good quality DNA from small samples of arthropod tissues generating low quantities of hazardous wastes. This new method was compared with another homemade protocol using phenol and other two commercial kits. The quality of DNA obtained was checked by spectrophotometer and evaluated by an AFLP assay. Low shearing DNA was obtained from all samples and the best readings were observed to DNA recollected with the new method. The AFLP assay indicated that DNA obtained with all methods were suitable for use in molecular biology techniques sensitive to contaminants. However, homemade protocols were more efficient in recollect DNA than commercial kits, without lose any quality of samples. Also, they were less time and fund consuming, with costs ten times cheaper than commercial kits. The quicker, less pollutant and cheaper protocol was the one described here (USD 0.52 per sample).

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Aqui, é apresentado um método rápido e eficiente para obtenção de DNA de boa qualidade a partir de pequenas amostras de tecidos de artrópodos, gerando pequenas quantidades de resíduos perigosos. Comparamos a eficiência do método com outro protocolo caseiro utilizando fenol e com dois kits comerciais. A qualidade do DNA obtido foi verificada em espectrofotômetro e avaliada por um ensaio de AFLP. Foi obtido DNA pouco fragmentado a partir de todas as amostras, mas as melhores leituras foram obtidas para o DNA extraído com o novo método. O ensaio de AFLP indicou que os DNAs obtidos estavam adequados para uso em técnicas de biologia molecular sensíveis a contaminantes. Porém, os protocolos caseiros foram mais eficientes em extrair DNA do que kits comerciais, sem perder nenhuma qualidade na pureza das amostras. Além disso, eles foram mais rápidos e baratos, chegando a custar dez vezes menos que os kits comerciais. O protocolo mais rápido, menos poluente e mais barato foi o descrito aqui (USD 0,52 por amostra).

RESUMO

Here, it is presented a rapid and efficient method to obtain good quality DNA from small samples of arthropod tissues generating low quantities of hazardous wastes. This new method was compared with another homemade protocol using phenol and other two commercial kits. The quality of DNA obtained was checked by spectrophotometer and evaluated by an AFLP assay. Low shearing DNA was obtained from all samples and the best readings were observed to DNA recollected with the new method. The AFLP assay indicated that DNA obtained with all methods were suitable for use in molecular biology techniques sensitive to contaminants. However, homemade protocols were more efficient in recollect DNA than commercial kits, without lose any quality of samples. Also, they were less time and fund consuming, with costs ten times cheaper than commercial kits. The quicker, less pollutant and cheaper protocol was the one described here (USD 0.52 per sample).

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Aqui, é apresentado um método rápido e eficiente para obtenção de DNA de boa qualidade a partir de pequenas amostras de tecidos de artrópodos, gerando pequenas quantidades de resíduos perigosos. Comparamos a eficiência do método com outro protocolo caseiro utilizando fenol e com dois kits comerciais. A qualidade do DNA obtido foi verificada em espectrofotômetro e avaliada por um ensaio de AFLP. Foi obtido DNA pouco fragmentado a partir de todas as amostras, mas as melhores leituras foram obtidas para o DNA extraído com o novo método. O ensaio de AFLP indicou que os DNAs obtidos estavam adequados para uso em técnicas de biologia molecular sensíveis a contaminantes. Porém, os protocolos caseiros foram mais eficientes em extrair DNA do que kits comerciais, sem perder nenhuma qualidade na pureza das amostras. Além disso, eles foram mais rápidos e baratos, chegando a custar dez vezes menos que os kits comerciais. O protocolo mais rápido, menos poluente e mais barato foi o descrito aqui (USD 0,52 por amostra).

RESUMO

Here, it is presented a rapid and efficient method to obtain good quality DNA from small samples of arthropod tissues generating low quantities of hazardous wastes. This new method was compared with another homemade protocol using phenol and other two commercial kits. The quality of DNA obtained was checked by spectrophotometer and evaluated by an AFLP assay. Low shearing DNA was obtained from all samples and the best readings were observed to DNA recollected with the new method. The AFLP assay indicated that DNA obtained with all methods were suitable for use in molecular biology techniques sensitive to contaminants. However, homemade protocols were more efficient in recollect DNA than commercial kits, without lose any quality of samples. Also, they were less time and fund consuming, with costs ten times cheaper than commercial kits. The quicker, less pollutant and cheaper protocol was the one described here (USD 0.52 per sample).

---

Aqui, é apresentado um método rápido e eficiente para obtenção de DNA de boa qualidade a partir de pequenas amostras de tecidos de artrópodos, gerando pequenas quantidades de resíduos perigosos. Comparamos a eficiência do método com outro protocolo caseiro utilizando fenol e com dois kits comerciais. A qualidade do DNA obtido foi verificada em espectrofotômetro e avaliada por um ensaio de AFLP. Foi obtido DNA pouco fragmentado a partir de todas as amostras, mas as melhores leituras foram obtidas para o DNA extraído com o novo método. O ensaio de AFLP indicou que os DNAs obtidos estavam adequados para uso em técnicas de biologia molecular sensíveis a contaminantes. Porém, os protocolos caseiros foram mais eficientes em extrair DNA do que kits comerciais, sem perder nenhuma qualidade na pureza das amostras. Além disso, eles foram mais rápidos e baratos, chegando a custar dez vezes menos que os kits comerciais. O protocolo mais rápido, menos poluente e mais barato foi o descrito aqui (USD 0,52 por amostra).

RESUMO

Here, it is presented a rapid and efficient method to obtain good quality DNA from small samples of arthropod tissues generating low quantities of hazardous wastes. This new method was compared with another homemade protocol using phenol and other two commercial kits. The quality of DNA obtained was checked by spectrophotometer and evaluated by an AFLP assay. Low shearing DNA was obtained from all samples and the best readings were observed to DNA recollected with the new method. The AFLP assay indicated that DNA obtained with all methods were suitable for use in molecular biology techniques sensitive to contaminants. However, homemade protocols were more efficient in recollect DNA than commercial kits, without lose any quality of samples. Also, they were less time and fund consuming, with costs ten times cheaper than commercial kits. The quicker, less pollutant and cheaper protocol was the one described here (USD 0.52 per sample).

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Aqui, é apresentado um método rápido e eficiente para obtenção de DNA de boa qualidade a partir de pequenas amostras de tecidos de artrópodos, gerando pequenas quantidades de resíduos perigosos. Comparamos a eficiência do método com outro protocolo caseiro utilizando fenol e com dois kits comerciais. A qualidade do DNA obtido foi verificada em espectrofotômetro e avaliada por um ensaio de AFLP. Foi obtido DNA pouco fragmentado a partir de todas as amostras, mas as melhores leituras foram obtidas para o DNA extraído com o novo método. O ensaio de AFLP indicou que os DNAs obtidos estavam adequados para uso em técnicas de biologia molecular sensíveis a contaminantes. Porém, os protocolos caseiros foram mais eficientes em extrair DNA do que kits comerciais, sem perder nenhuma qualidade na pureza das amostras. Além disso, eles foram mais rápidos e baratos, chegando a custar dez vezes menos que os kits comerciais. O protocolo mais rápido, menos poluente e mais barato foi o descrito aqui (USD 0,52 por amostra).