LARGE-SCALE PRODUCTION OF PALINDROME DNA FRAGMENTS

Our structural studies of nucleosomes necessitated the production of o ver 100 mg of a 146-bp perfect palindrome DNA for use in the reconstit ution of perfectly symmetrical nucleosome core particles for detailed Xray crystallographic analysis. The propagation of palindromic DNA seq uences by bacterial culture is hindered by the instability of these se quences 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 coil, not all palindrome-containing plasmi ds are faithfully maintained by these strains. The production of large quantities of palindrome DNA can therefore be extremely difficult. Af ter trying several approaches, we were able to develop a reliable proc edure for production of large quantities of palindrome DNA that involv es production of plasmid containing multiple copies of the repeating u nit of the palindrome which are isolated by restriction digestion and ligated in vitro to form the palindrome DNA. The procedure has resulte d in the production of over 20 mg of a 146-bp DNA fragment in 2 weeks. (C) 1995 Academic Press, Inc.