Aislamiento de ADN de alta calidad en Psidium guajava L. para estudios genómicos
Fecha
2021
Tipo
artículo original
Autores
Sánchez Barrantes, Elodia María
Mora Newcomer, Eric
Barrantes Santamaría, Walter
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Resumen
Introducción. La guayaba es uno de los principales frutales de la familia Mirtáceas por su alto valor nutricional.
Es originario de América tropical y los principales países productores son: México, India, Brasil y Tailandia. El interés
generado en los últimos años por el mejoramiento genético de este cultivo, ha propiciado el empleo de herramientas
moleculares que permitan determinar la variabilidad genética y seleccionar genes de interés agronómico de una
forma rápida y confiable. Sin embargo, el aislamiento de ADN de alta pureza es un requisito previo para poder
emplear las técnicas moleculares de última generación. Objetivo. Aislar ADN genómico (ADNg) de alta calidad de
guayaba, en cantidad e integridad adecuados. Materiales y métodos. El experimento se realizó en el Laboratorio de
Biología Molecular de la Estación Experimental Agrícola Fabio Baudrit Moreno, entre enero y diciembre del 2019.
Se compararon tres métodos diferentes para aislar ADN: Promega (ReliaPrep™ gDNA Tissue Miniprep Kit), Qiagen
(DNeasy Plant Mini Kit) y CTAB (Doyle y Doyle, 1990) con modificaciones. Para la extracción de ANDg se utilizó
material fresco y liofilizado de hojas jóvenes de guayaba (Psidium guajava L.; 2n = 22). Para determinar el mejor
método se midieron la calidad, cantidad e integridad del ADNg de cada uno. Resultados. Se logró obtener ADNg
con los tres métodos evaluados, los mejores resultados en cantidad de ADNg (ng µl-1) se obtuvieron con el material
liofilizado y con el método CTAB. Los métodos CTAB y Qiagen mostraron mayor grado de pureza (relaciones
A260/280 con valores óptimos) en comparación con el método Promega. Conclusión. Se logró obtener ADNg en
cantidad, calidad e integridad adecuada. Esto se logró con base en extracciones en tejido liofilizado de hojas jóvenes
y con el método de extracción del kit de Qiagen.
Introduction. Guava is one of the main fruit trees of the Mirtáceas family for its high nutritional value. It is originally from tropical America and the main producing countries are: Mexico, India, Brazil, and Thailand. The interest generated in recent years by the genetic improvement of this crop, has led to the use of molecular tools that allow determining genetic variability and selecting genes of agronomic interest in a fast and reliable way. However, the isolation of highpurity DNA is a prerequisite for the use of state-of-the-art molecular techniques for genetic analysis. Objective. Isolate high-quality genomic DNA (gDNA) from guava, in adequate quantity and integrity. Materials and methods. The experiment was carried out in the Molecular Biology Laboratory of the Estacion Experimental Agricola Fabio Baudrit Moreno, between January and December 2019. Three different methods for isolating DNA were compared: Promega (ReliaPrep™ gDNA Tissue Miniprep Kit), Qiagen (DNeasy Plant Mini Kit), and CTAB (Doyle and Doyle, 1990) with modifications. For the gDNA extraction, fresh and lyophilized of young leaves of guava (Psidium guajava L; 2n = 22) were used. To determine the best method, the quality, quantity, and integrity of the gDNA were measured for each method. Results. The gDNA was obtained with the three evaluated methods. The best results in terms of gDNA quantity (ng µl-1) were obtained with the lyophilized material and with the CTAB method (Doyle and Doyle). The CTAB (Doyle and Doyle) and Qiagen methods showed a higher degree of purity (A260 / 280 ratios with optimal values) compared to the Promega method. Conclusion. The gDNA was obtained in adequate quantity, quality, and integrity. This was achieved based on extractions in lyophilized young leaf tissue and with the extraction method of the Qiagen kit.
Introduction. Guava is one of the main fruit trees of the Mirtáceas family for its high nutritional value. It is originally from tropical America and the main producing countries are: Mexico, India, Brazil, and Thailand. The interest generated in recent years by the genetic improvement of this crop, has led to the use of molecular tools that allow determining genetic variability and selecting genes of agronomic interest in a fast and reliable way. However, the isolation of highpurity DNA is a prerequisite for the use of state-of-the-art molecular techniques for genetic analysis. Objective. Isolate high-quality genomic DNA (gDNA) from guava, in adequate quantity and integrity. Materials and methods. The experiment was carried out in the Molecular Biology Laboratory of the Estacion Experimental Agricola Fabio Baudrit Moreno, between January and December 2019. Three different methods for isolating DNA were compared: Promega (ReliaPrep™ gDNA Tissue Miniprep Kit), Qiagen (DNeasy Plant Mini Kit), and CTAB (Doyle and Doyle, 1990) with modifications. For the gDNA extraction, fresh and lyophilized of young leaves of guava (Psidium guajava L; 2n = 22) were used. To determine the best method, the quality, quantity, and integrity of the gDNA were measured for each method. Results. The gDNA was obtained with the three evaluated methods. The best results in terms of gDNA quantity (ng µl-1) were obtained with the lyophilized material and with the CTAB method (Doyle and Doyle). The CTAB (Doyle and Doyle) and Qiagen methods showed a higher degree of purity (A260 / 280 ratios with optimal values) compared to the Promega method. Conclusion. The gDNA was obtained in adequate quantity, quality, and integrity. This was achieved based on extractions in lyophilized young leaf tissue and with the extraction method of the Qiagen kit.
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Palabras clave
Guayaba, Métodos de extracción de ADN, Integridad ADN, Cuantificación de ADN, Liofilización, Secuenciación, Guava, DNA extraction methods, DNA integrity, DNA quantification, Lyophilization, Sequencing