DNA was fragmented using restriction endonucleases() from Boehringer, Gibco BRL (Gaitersburg, MD), or New England Biolabs (Beverly, MA), with buffers and according to the specifications of the supplier. In the case of multiple digestion with enzymes that require different buffers, the reactions were carried out consecutively, inactivating the enzymes in between by a phenol/chloroform/isoamyl alcohol (25/24/1) extraction, and followed by gel permeation chromatography over a Sephadex G50 column (Pharmacia) to desalt the samples. To analyse restriction fragments, DNA products were separated by agarose gel electrophoresis (agarose at concentrations of 0.7 up to 2% (w/v)) in TAE electrophoresis buffer (40 mM Tris.HCl pH 7.2, 500 mM sodium acetate, 50 mM EDTA). After staining with ethidium bromide, DNA bands were visualized by UV illumination (Sambrook et al., 1989).
Isolation and purification of DNA restriction fragments after semi-preparative gel electrophoresis was done with QiaEx or QIAquick gel extraction kits (Qiagen). In the first step of these procedures, the agarose matrix, containing the desired DNA fragments, was solubilized; subsequently, DNA fragments were selectively bound onto silica-gel particles or silica-gel membranes, respectively. All steps were performed according to the specifications of the supplier.
Ligation of compatible ends of DNA was catalyzed by T4 DNA ligase (Boehringer; Promega) during an overnight incubation at 16°C. Synthetic DNA fragments were phosphorylated with T4 polynucleotide kinase. Dephosphorylation of restriction fragments was performed with calf intestine phosphatase. All these reactions were carried out with buffers of the enzyme suppliers.