Sequential amplification of cloned DNA as tandem multimers using class-IIS restriction enzymes

In order to make high-copy-number multimers of DNA fragments in a tandem unit, two different gene amplification vectors (pSK9 and pBBS1) were developed. Two identical class-IIS restriction enzyme sites (BspMI for pSK9 and BbsI for pBBSI) were inversely oriented in each vector with the same cut site, creating asymmetric and complementary cohesive ends (5'-CCCC and 5'-GGGG). Multimers were made by: (i) cloning a target DNA into the class-IIS restriction enzyme cut site of each vector; (ii) excision of the monomeric insert by digestion with the class-IIS restriction enzyme; (iii) isolation of the fragments; (iv) self-ligation of the fragments; (v) cloning into the original vector digested with the class-IIS restriction enzyme; and (vi) repeating steps (i) through (v) to generate higher-order multimers. Various-sized multimers of a 93-bp DNA fragment encoding magainin, an antimicrobial peptide, were obtained with the gene amplification vector, pBBS1. Larger multimers, up to about 108 copies, were constructed from the monomer by the sequential amplification procedure. Of six different Escherichia coli hosts examined for the stability of multimers, the multimers were the most stable in E. coli D1210. The gene amplification vector system described here is very efficient and can be applied in the construction of tandem multimers of any kind of DNA, as long as the cloned DNA does not contain the cut site of the class-IIS restriction enzyme to be utilized.