Eyers M, Edwards K, Schuch W
Plant Biotechnology Section, ICI Seeds, Bracknell, UK.
Gene. 1992 Nov 16;121(2):195-201. doi: 10.1016/0378-1119(92)90122-6.
We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded protoplasts of a sugar beet (Beta vulgaris L.) cell suspension line, Ar+. This library contains 15,000 clones with an average insert size of 140 kb. Based on a sugar beet haploid genome size of 1.1 x 10(3) Mb it should represent approximately two haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. High-density filters, each containing 864 YAC clones, have been screened in colony hybridisation experiments. In this way, sugar beet YAC clones have been identified containing chloroplast DNA, mitochondrial DNA and two satellite DNAs. Ten pools of DNA from 1500 individual YAC clones have also been prepared for rapid PCR screening of the library. Using this approach, in combination with colony hybridisation, we have been able to isolate sugar beet YAC clones containing a transcribed low-copy-number gene.
我们利用从甜菜（Beta vulgaris L.）细胞悬浮系Ar + 的琼脂糖包埋原生质体制备的高分子量DNA构建了一个酵母人工染色体（YAC）文库。该文库包含15000个克隆，平均插入片段大小为140 kb。基于甜菜单倍体基因组大小为1.1×10³ Mb，它应该代表大约两个单倍体基因组当量。该文库在一式两份的微量滴定板中以有序阵列形式排列。在菌落杂交实验中筛选了高密度滤膜，每个滤膜包含864个YAC克隆。通过这种方式，已鉴定出含有叶绿体DNA、线粒体DNA和两种卫星DNA的甜菜YAC克隆。还制备了来自1500个单个YAC克隆的10组DNA池，用于对文库进行快速PCR筛选。使用这种方法，并结合菌落杂交，我们已经能够分离出含有转录的低拷贝数基因的甜菜YAC克隆。
