e109 Nucleic Acids Research, 2005, Vol. 33, No. 12 point mutations, deletions, insertions and substitutions at any place in the target genes as required. MATERIALS AND METHODS PCR conditions An aliquot of 20 pmol of primers and 50 ng of template DNA were used to obtain N- and C-terminal fragments by PCR with 0.5 U Pfx polymerase (Invitrogen Corp., Carlsbad, CA) in 25 ml reaction mixture. Cycling parameters for PCR were 15 cycles, consisting of 1 min at 95 C for denaturing, 2 min at 55 C for annealing, 3 min at 68 C for extension and 5 min at 68 C for ﬁnal extension. BP in vitro recombination BP recombination was according to the industrial manual with a half scale. Our condition is as follows: PCR products con- taining N- and C-terminal fragments (2.5 ml each) and 150 ng pDONR 201 were combined in a 10 ml reaction mixture con- taining 1 ml BP clonase (Invitrogen Corp.). It is not necessary to purify PCR products. The mixture was incubated at 25 C for 16 h. Transformation After the reaction was stopped by proteinase K, 2.5 ml of reaction mixture was mixed with 25 ml DH5a competent cell (Invitrogen Corp.) for transformation. The procedure was according to the industrial manual. All cells were spread on Luria–Bertani (LB) plate containing 50 mg/ml Kanamycin or 25 mg/ml Zeocin. RESULTS Experimental design The general outline of N- and C-terminal Gateway cloning method for chimeragenesis is illustrated in Figure 1. N- and C-terminal fragments were obtained by PCR with attB attached primers and internal gene primers. Internal gene pri- mers were designed to create a homologous region at the 30 end of N-terminal and 50 of C-terminal fragments. Symbols a and b in Figure 1 indicate gene speciﬁc sequences of joining sites of the 30 end of N- and 50 end of C-terminal fragments, respect- ively. These fragments were then introduced into a Donor vector (pDONR) by BP recombination (without any puriﬁca- tion steps). E.coli was then transformed with the linear plasmid product. The desired, mutated clone was obtained after the linear plasmid was circularized in the E.coli cell utilizing the homologous sequences at its ends. The length of homologous sequences required for plasmid circularization in E.coli To address the question of the minimal length of homologous sequences required for this cloning method, we prepared ﬁve attB1-N-terminal fragments containing various lengths of homology when paired with a single attB2-C-terminal. Five attB1-N-terminal fragments, N930, N940, N959, N998 and N1053, and one attB2-C-terminal fragment, C709, were produced by PCR. The length of each attB1-N- and PAGE 2 OF 6 Figure 1. Outline of chimeragenesis with Gateway technology. For details see text. attB2-C-terminal fragment was 930, 940, 959, 998, 1053 and 709 bp, respectively. Five attB1-N-terminal fragments had 10, 20, 39, 78 and 133 bp homologous sequences to attB2-C-terminal fragment at their ends. The design of PCR and products used for the BP reaction are shown in Figure 2. Each attB1-N-terminal fragment, attB2-C-terminal frag- ment and pDONR201 were mixed at a molar ratio of 5:5:1 and BP recombination was carried out. After stopping the reaction with proteinase K, linear DNA samples were directly introduced into E.coli DH5a (recA strain). The number of colonies obtained from 37.5 ng vector DNA and the efﬁciency of clone recovery for each condition are shown in Table 1. Even 10 bp homologous region gave 25 colonies. We ana- lyzed the sequences of the junction region between the N- and C-terminal fragment to check the ﬁdelity of homologous recombination in the recA strain. None of the six clones sequenced from each condition showed unexpected sequences in the junction region. This indicated that a 10 bp homologous sequence was sufﬁcient for correct homologous recombination at the ends of the plasmid, although the number of clones obtained was smaller than with other conditions. The effect of homologous sequence in the target gene Some genes contain tandem repeats or short homologous sequences. We wanted to determine the effect of such