van Burik J A, Schreckhise R W, White T C, Bowden R A, Myerson D
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, USA.
Med Mycol. 1998 Oct;36(5):299-303.
Filamentous fungi have a sturdy cell wall which is resistant to the usual DNA extraction procedures. We determined the DNA extraction procedure with the greatest yield of high quality fungal DNA and the least predilection for cross-contamination of equipment between specimens. Each of six extraction methods was performed using Aspergillus fumigatus hyphae. The six methods were: (1) glass bead pulverization with vortexing; (2) grinding with mortar and pestle followed by glass bead pulverization; (3) glass bead pulverization using 1% hydroxyacetyl trimethyl ammonium bromide (CTAB) buffer in a water bath sonicator; (4) water bath sonication in CTAB buffer; (5) grinding followed by incubation with CTAB; and (6) lyticase enzymatic cell lysis. Genomic DNA yields were measured by spectrophotometry and by visual reading of 2% agarose gels, with shearing assessed by the migration of the DNA on the gel. Genomic fungal DNA yields were highest for Method 1, followed by Methods 5 approximately = to 2 >3 approximately = to 4 approximately = to 6. Methods 2 and 5, both of which involved grinding with mortar and pestle, led to shearing of the genomic DNA in one of two trials each. We conclude that the use of glass beads with extended vortexing is optimal for extraction of microgramme amounts of DNA from filamentous fungal cultures.
丝状真菌具有坚固的细胞壁，能抵抗常规的DNA提取程序。我们确定了能获得最高产量高质量真菌DNA且对样本间设备交叉污染倾向最小的DNA提取程序。使用烟曲霉菌丝对六种提取方法分别进行了操作。这六种方法分别是：（1）玻璃珠研磨并涡旋振荡；（2）用研钵和研杵研磨后再进行玻璃珠研磨；（3）在水浴超声仪中使用1%十六烷基三甲基溴化铵（CTAB）缓冲液进行玻璃珠研磨；（4）在CTAB缓冲液中进行水浴超声处理；（5）研磨后与CTAB孵育；（6）用溶菌酶进行酶促细胞裂解。通过分光光度法以及对2%琼脂糖凝胶的目视读数来测定基因组DNA产量，并通过DNA在凝胶上的迁移情况评估DNA的剪切。方法1获得的真菌基因组DNA产量最高，其次是方法5约等于方法2大于方法3约等于方法4约等于方法6。方法2和方法5都涉及用研钵和研杵研磨，在各自的两次试验中，有一次导致了基因组DNA的剪切。我们得出结论，长时间涡旋振荡使用玻璃珠对于从丝状真菌培养物中提取微克量的DNA是最佳的。
