Albertsen H M, Abderrahim H, Cann H M, Dausset J, Le Paslier D, Cohen D
Centre d'Etude du Polymorphisme Humain, Paris, France.
Proc Natl Acad Sci U S A. 1990 Jun;87(11):4256-60. doi: 10.1073/pnas.87.11.4256.
Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, we performed a series of preliminary experiments aimed at developing a suitable protocol. We found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes.
在构建包含非常大的人类DNA片段的酵母人工染色体（YAC）文库之前，我们进行了一系列旨在开发合适方案的初步实验。我们发现YAC插入片段大小与转化效率之间呈反比关系。发现YAC插入片段内偶尔会发生重排，导致克隆稳定的内部缺失或克隆不稳定的大小变化。开发了一种方案，用于从部分限制性消化产物中制备性电泳富集高分子量人类DNA片段，并在琼脂糖中与YAC载体连接。已从爱泼斯坦-巴尔病毒转化的人类淋巴母细胞系的大片段DNA构建了一个YAC文库。该文库目前包含50,000个克隆，其中95%的大小大于250千碱基对。根据132个随机分离的克隆计算，该文库的平均YAC大小为430千碱基对。因此，该文库包含大约相当于七个单倍体人类基因组的DNA。
