Rudi K, Kroken M, Dahlberg O J, Deggerdal A, Jakobsen K S, Larsen F
University of Oslo, Norway.
Biotechniques. 1997 Mar;22(3):506-11. doi: 10.2144/97223rr01.
A magnetic bead-based system for DNA isolation utilizing monodisperse beads was tested with the aim of producing a general approach for PCR-ready DNA. This commercially available system was originally designed for isolating PCR-ready DNA from human whole blood. We tested diverse organisms belonging to the major groups: bacteria, fungi, algae, vascular plants and vertebrates. Optimization of sample amounts and lysis conditions was done using several types of tissue (fish epithelium, plant leaves, mammalian liver and muscle tissues, fungal fruit-bodies and mycelium). The standard lysis conditions used for blood could be applied with good results for most bacteria, algae and vertebrates, while plant leaves and fungal fruit-bodies had to be mechanically broken to obtain proper lysis. For vascular plants and some cyanobacteria, lysis by heating to 65 degrees C gave better DNA yields than standard lysis at room temperature. In all cases, DNA suitable for PCR was prepared in less than 30 min. The PCR products yielded 350 to 500 bases of DNA sequence (99% accurate) by direct manual or automated sequencing.
为了开发一种通用的制备适用于PCR的DNA的方法，对一种利用单分散磁珠的DNA分离磁珠系统进行了测试。这种市售系统最初设计用于从人全血中分离适用于PCR的DNA。我们测试了分属于主要类群的多种生物：细菌、真菌、藻类、维管植物和脊椎动物。使用几种类型的组织（鱼上皮、植物叶片、哺乳动物肝脏和肌肉组织、真菌子实体和菌丝体）对样品量和裂解条件进行了优化。用于血液的标准裂解条件对大多数细菌、藻类和脊椎动物适用且效果良好，而植物叶片和真菌子实体必须进行机械破碎才能实现充分裂解。对于维管植物和一些蓝细菌，加热至65℃进行裂解比在室温下进行标准裂解能获得更高的DNA产量。在所有情况下，均可在不到30分钟的时间内制备出适用于PCR的DNA。通过直接手动或自动测序，PCR产物产生了350至500个碱基的DNA序列（准确率99%）。
