A crush on small fungi: an efficient and quick method for obtaining DNA from minute ascomycetes

1. We have developed a reliable technique for extracting DNA from single microscopic fungal thalli, including efficient cell disruption and transfer of cell content for subsequent polymerase chain reaction (PCR). The technique was primarily developed for members of the ascomycete order Laboulbeniales, which are minute fungi with tough cell walls that are exceedingly difficult to disrupt with standard extraction techniques. 2. Our method makes routine amplification of DNA from single thalli possible, even from small species or poorly developed individuals. DNA release is accomplished in an entirely mechanical manner using an arbor press fitted with custom-made components. This approach has eliminated additional treatment such as laborious freeze-thaw cycles, enzymes, or lysing agents. 3. The overall PCR success rate of 89% is comparable to or better than alternative protocols that make use of substantially larger amounts of fungal tissue. From 97% of the successful PCRs a total of 156 s...1. 我们开发了一种可从单株显微真菌菌体中提取DNA的可靠技术，该技术涵盖高效细胞裂解与细胞内容物转移流程，可用于后续聚合酶链式反应（polymerase chain reaction, PCR）实验。本技术最初针对子囊菌纲虫囊菌目（Laboulbeniales）类群开发，这类真菌体型微小且细胞壁坚韧，采用常规提取技术极难实现细胞裂解。 2. 本方法可实现单株菌体DNA的常规扩增，即便针对小型物种或发育不完全的个体亦可行。DNA释放全程采用纯机械方式完成，通过加装定制组件的手动台式压床实现。该方案无需额外处理步骤，免去了繁琐的冻融循环、酶解或裂解试剂等操作。 3. 本方法的PCR总体成功率达89%，与需使用大量真菌组织的替代实验方案相比相当甚至更优。在97%的成功PCR扩增反应中，我们共获取了总计156个[原文未表述完整]。