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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Publications at this Location » Publication #62886


item Tooley, Paul
item Carras, Marie

Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: The fungus Phytophthora infestans causes potato blight and DNA studies are needed to identify strains of the fungus and investigate its genetic makeup. Existing methods for DNA extraction from this fungus are time-consuming and involve use of toxic chemicals such as phenol. Ultra-pure DNA and DNA of very high molecular weight are needed for construction of genomic libraries is currently produced using an even more lengthy procedure. This paper describes an adaptation of a commercially available DNA extraction procedure which reduces the time necessary for DNA extraction from P. infestans, and yields ultra-pure DNA of high molecular weight

Technical Abstract: High molecular weight DNA of Phytophthora infestans was extracted using a modification of the commercial QIAGEN column procedure. Both a "maxi" and "mini" procedure are described. The "maxi" procedure utilizes a QIAGEN-tip 500 column and yielded over 300 Hg of DNA from 500 mg lyophilized fungal tissue. The "mini" procedure utilizes a QIAGEN-tip 20 column and yielded 5-10 Hg DNA from 60 mg lyophilized tissue. When fungal protoplasts were used as starting material from ca. 9 g fresh weight of fungal tissue, nearly 500 Hg of DNA in the size range of 20-200 kb was obtained, suitable for construction of a lambda genomic library. The modified QIAGEN method can replace the more tedious, time-consuming, and expensive cesium chloride density gradient centrifugation method for extraction of ultra-pure fungal DNA from P. infestans and perhaps other filamentous fungi