Rapid and Efficient Method for the Extraction of Fungal and Oomycetes Genomic DNA

نویسندگان

  • Ajay Kumar Mishra
  • Kamal Sharma
  • Raj Shekhar Misra
چکیده

An improved protocol for the isolation of high-quality DNA from fungi is described. The method involves inactivating proteins by SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high molecular weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous chemical such as phenol and as many as 50 samples can be processed per day. The purity of isolated genomic DNA was confirmed by means of spectrophotometer analysis (A260/280 ratio of 1.80-1.96), indicated a minimal presence of contaminating metabolites. The method yielded 0.55-0.92 μg DNA mg freeze dried mycelia, when tested on three fungal species Fusarium solani, Colletotrichum capsici, and Rhizoctonia solani and two oomycetes Phytophthora colocasiae and Pythium aphanidermatum. The DNA was completely digested with EcoRI and HindIII. PCR-based technique such as random amplification of polymorphic DNA (RAPD), showed the DNA’s compatibility with downstream applications. _____________________________________________________________________________________________________________

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تاریخ انتشار 2009