Directional PCR cloning of multiple repeat sequences.

The tandem ligation of DNA sequences within plasmid vectors is hindered by the inability to force the orientation of subsequently ligated DNA fragments and the inherent instability of repeated sequences when grown in bacterial hosts. Improvements have been made in the latter by the introduction of several E. coli strains by commercial suppliers (STBL2; Life Technologies, Gaithersburg, MD, USA or SURE® and SURE2; Stratagene, La Jolla, CA, USA). These contain inactivating mutations in genes responsible for homologous recombination, DNA repair and/or restriction modification and as such, can maintain plasmids containing both inverted and direct repeated DNA sequences (2,6,7). However, since the ligation of multiple sequences in a defined context is hindered by the determination of the fragment orientation, I have developed a rapid polymerase chain reaction (PCR)/ligation protocol for the sequential cloning of DNA fragments. This approach relies on the compatibility of the 5′ overhangs generated by the restriction enzymes BamHI (G↓GATCC) and BglII (A↓GATCT). These two enzymes were chosen for several reasons: (i) they create compatible 5′ overhangs that are crucial for the success of this procedure; (ii) they both cleave under similar ionic and reaction conditions (React 3; Life Technologies) at 37°C; (iii) they cleave relatively infrequently in genomic DNA; (iv) only 2 bp of an extra sequence at either end of their recognition site are required for >90% cleavage (5); and (v) they are readily available and inexpensive. Directional ligation. The schematic for the procedure for multiple ligation is indicated in Figure 1. PCR primers containing sequences complementary to the desired target are designed containing BamHI and BglII sites at the 5′ and 3′ ends, respectively. Following amplification (Figure 1A), the product is ligated into pSL1190 (Pharmacia Biotech, Piscataway, NJ, USA) restricted with BamHI and BglII, and the product is transformed into E. coli STBL2. Since the fragment can be ligated in both orientations (due to the GATC overhang generated by both enzymes), the sequence context of the insert must be determined by digestion with both BamHI and BglII. The ligation of a BamHI cohesive end to a BglII cohesive end and vice versa generate sequences that cannot be cleaved by either enzymes. Hence, two species of DNA will result from the ligation, one that will regenerate the original PCR product and one that will not be cleaved following restriction by the two enzymes. One copy of the desired sequence has therefore been cloned (Figure 1B). Tandem ligation of multiple fragments. To add additional copies, the plasmid is digested with BglII, ligated with the original fragment and bacteria retransformed (Figure 1C). The subsequently ligated fragment can be orien-