Gingrich J C, Boehrer D M, Basu S B
Genetic Systems Division, Bio-Rad Laboratories, Hercules, CA 94547, USA.
Biotechniques. 1996 Jul;21(1):99-104. doi: 10.2144/96211st04.
We demonstrate an alternative method for the generation of random subclones for large-scale shotgun human DNA sequencing projects. Miniprep DNA from a human cosmid clone was partially digested with CviJI, size-fractionated by agarose gel electrophoresis and cloned into bacteriophage M13. A library consisting of 10(5) subclones of 1.1 kb average size was recovered from one gel fraction containing approximately 300 ng of partially digested DNA. DNA sequences from an initial 103 subclones demonstrate that 100 of the subclones cover 90% of the cosmid, which is close to the 92% expected if the subclones were generated at random. DNA sequences from three of the subclones did not match the cosmid sequences, establishing that miniprep DNA can be used for library construction with little concern for contamination with genomic E. coli DNA. The use of CviJI to generate random DNA fragments thus offers a simple alternative to other commonly used fragmentation methods, such as shearing or sonication, for the generation of random sublibraries for large-scale human shotgun DNA sequencing projects.
我们展示了一种为大规模鸟枪法人类DNA测序项目生成随机亚克隆的替代方法。从人黏粒克隆中提取的小量制备DNA用CviJI进行部分酶切，通过琼脂糖凝胶电泳进行大小分级分离，然后克隆到噬菌体M13中。从一个含有约300 ng部分酶切DNA的凝胶片段中获得了一个由10⁵个平均大小为1.1 kb的亚克隆组成的文库。最初10³个亚克隆的DNA序列表明，其中100个亚克隆覆盖了黏粒的90%，这接近如果亚克隆是随机生成时预期的92%。三个亚克隆的DNA序列与黏粒序列不匹配，这表明小量制备DNA可用于文库构建，而无需过多担心被基因组大肠杆菌DNA污染。因此，使用CviJI生成随机DNA片段为大规模人类鸟枪法DNA测序项目生成随机亚文库提供了一种简单的替代方法，可替代其他常用的片段化方法，如剪切或超声处理。
