Palmer E L, Gewiess A, Harp J M, York M H, Bunick G J
University of Tennessee/Oak Ridge Graduate School of Biomedical Sciences, USA.
Anal Biochem. 1995 Oct 10;231(1):109-14. doi: 10.1006/abio.1995.1509.
Our structural studies of nucleosomes necessitated the production of over 100 mg of a 146-bp perfect palindrome DNA for use in the reconstitution of perfectly symmetrical nucleosome core particles for detailed X-ray crystallographic analysis. The propagation of palindromic DNA DNA sequences by bacterial culture is hindered by the instability of these sequences during bacterial replication and recombination. While the loss of some palindrome sequences can be eliminated by the use of sbcB or sbcC mutants of Escherichia coli, not all palindrome-containing plasmids are faithfully maintained by these strains. The production of large quantities of palindrome DNA that involves production of plasmid containing multiple copies of the repeating unit of the palindrome which are isolated by restriction digestion and ligated in vitro to form the palindrome DNA. The procedure has resulted in the production of over 20 mg of a 146-bp DNA fragment in 2 weeks.
我们对核小体的结构研究需要制备超过100毫克的146碱基对完美回文DNA，用于重建完全对称的核小体核心颗粒，以进行详细的X射线晶体学分析。细菌培养对回文DNA序列的扩增存在阻碍，因为这些序列在细菌复制和重组过程中不稳定。虽然使用大肠杆菌的sbcB或sbcC突变体可以消除一些回文序列的丢失，但并非所有含回文序列的质粒都能被这些菌株忠实地维持。大量回文DNA的制备涉及构建含有多个回文重复单元拷贝的质粒，通过限制性消化进行分离，并在体外连接形成回文DNA。该方法在两周内已成功制备出超过20毫克的146碱基对DNA片段。
