Macas J, Gualberti G, Nouzová M, Samec P, Lucretti S, Dolezel J
Institute of Plant Molecular Biology, Branisovská, Czech Republic.
Chromosome Res. 1996 Nov;4(7):531-9. doi: 10.1007/BF02261781.
Recombinant DNA libraries were constructed for seven chromosome types isolated from two translocation lines of field bean (Vicia faba L.) with reconstructed karyotypes. The chromosomes were selected so that the set of libraries covers the whole V. faba genome more than once. Individual chromosome types were highly purified by flow sorting, and their DNA was amplified by degenerate oligonucleotide-primed (DOP) polymerase chain reaction (PCR) and cloned into a plasmid vector. The choice of restriction site present in PCR primer and refinement of cloning protocol resulted in high cloning efficiency and allowed generation of libraries consisting of about 10(5) clones from 250 or 1000 sorted chromosomes. The insert size ranged between 50 and 2200 bp and the mean length estimated in individual libraries varied between 310 and 487 bp. Hybridization of cloned fragments with labelled genomic DNA showed that about 60% of inserts represented unique or low-copy sequences. The suitability of the libraries for genome mapping was demonstrated by isolation of clones containing microsatellite motifs.
为从具有重建核型的蚕豆（Vicia faba L.）两个易位系中分离出的七种染色体类型构建了重组DNA文库。选择这些染色体是为了使文库集能多次覆盖整个蚕豆基因组。通过流式细胞分选对单个染色体类型进行高度纯化，其DNA通过简并寡核苷酸引物（DOP）聚合酶链反应（PCR）进行扩增，并克隆到质粒载体中。PCR引物中存在的限制性酶切位点的选择以及克隆方案的优化导致了高克隆效率，并允许从250个或1000个分选的染色体中产生由约10⁵个克隆组成的文库。插入片段大小在50至2200 bp之间，各个文库中估计的平均长度在310至487 bp之间变化。克隆片段与标记的基因组DNA的杂交表明，约60%的插入片段代表独特或低拷贝序列。通过分离含有微卫星基序的克隆证明了文库对基因组作图的适用性。
