Método para la extracción de ADN cloroplastídico de Bouteloua gracilis como herramienta para aplicaciones moleculares
Method of chloroplast DNA extraction from Bouteloua gracilis as a tool for molecular applications
DOI:
https://doi.org/10.54167/tch.v5i3.691Palabras clave:
Análisis molecular, clonación de genes, mapa genético, transferencia horizontal de genesResumen
La manipulación genética del genoma de cloroplasto esta a la vanguardia en investigación biotecnológica. El pasto B. gracilis es un modelo para el estudio de estrés hídrico y oxidativo, capacidades relacionadas al cloroplasto. Es necesario tener un método que facilite la obtención de ADN de buena calidad, particularmente cuando se refiere al ADN de cloroplasto de B. gracilis. La implementación de un método para la extracción de cpADN se logró probando diferentes estrategias de extracción de cpADN reportados en la literatura y combinando las etapas más apropiadas para B. gracilis. La incorporación de un paso adicional del lavado con CTAB permitió la recuperación de cpDNA enriquecido, el cual se puede utilizar en el desarrollo de hermanitas moleculares. Debido la implementación de un protocolo de extracción también fue posible obtener una cantidad considerable de cpADN de B. gracilis. El cpADN fue digerido con varias enzimas de restricción y los fragmentos resultantes fueron analizados por Southern blot con las sondas de los genes de rADN 16S y 23S. Abstract Genetic manipulation of the chloroplast genome is at the forefront of biotechnology research. B. gracilis is a model for the study of water stress and oxidative capacities related to the chloroplast. It is requires a method to facilitate the obtaining of good quality DNA, particularly when it comes to chloroplast DNA of B. gracilis. The implementation of a method for extracting cpDNA was achieved by assaying different strategies cpDNA extraction reported in the literature and combining the most appropriate stage for B. gracilis for cpDNA extraction. The incorporation of an additional step of washing with CTAB after lysis of chloroplasts, allowed the recovery of enriched cpDNA which can be used in the development of molecular tools. Due to the implementation of an extraction protocol was also possible to obtain a considerable amount of B. gracilis cpDNA. The cpDNA was digested with several restriction enzymes and the resulting fragments were analyzed by Southern blot with probes of genes rADN 16S and 23S.Descargas
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