Song J, Dong F, Lilly J W, Stupar R M, Jiang J
Department of Horticulture, University of Wisconsin-Madison, 53706, USA.
Genome. 2001 Jun;44(3):463-9.
The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed.
作为细菌人工染色体(BAC)对大片段DNA进行克隆和扩增,已成为基因组研究中的一项重要技术。BAC克隆在宿主大肠杆菌中高度稳定,这是相对于酵母人工染色体(YAC)的一个主要优势,在YAC中,重组诱导的不稳定性是一个主要缺点。在此,我们报告含有串联重复DNA元件的BAC克隆不稳定,在常规文库维护和DNA制备过程中会发生剧烈缺失。在来自高粱、水稻和马铃薯的三个BAC克隆中观察到不稳定性,每个克隆都含有不同的串联重复序列。分别在生长24小时和120小时后,来自一个含有5S核糖体RNA基因的水稻BAC克隆的单菌落中,多达46%和74%出现了插入缺失。我们还证明,BAC插入重排可能发生在文库构建和复制的早期阶段。因此,最小生长法可能无法避免此类克隆的不稳定性问题。文中讨论了BAC不稳定性对基因组研究的影响。