PAGE 5 OF 6 (a) (b) (c) Figure 4. Summary of HTP N- and C-terminal cloning. (a) Gel showed N- and C-terminal fragments used for cloning and inserted fragments of clones. Symbols N and C indicate N- and C-terminal fragments, and I indicates insert fragments amplified by PCR. The dots indicate the position of missing fragments; green for N- or C-terminal; and yellow for right insert fragments. (b and c) Number of obtained colonies and the rate of right clone. Nucleic Acids Research, 2005, Vol. 33, No. 12 e109 sites were separated from each other, the resultant clone by the N- and C-terminal method was a deletion clone that lacked the sequence between the two priming sites. Using mutated primer and primers containing additional sequences at the 50 end of oligo, DNA produced a point mutant and insertion clone, respectively. The combination of deletion and insertion procedures gave a substitution clone. We constructed a point mutation, 120 bp deletion, 80 bp insertion and 80 bp substi- tution clones with 1.5 kb cDNA and evaluated these mutant clones by sequencing. We checked six clones of each mutant, and all clones showed the designed sequence in the whole region of the gene. DISCUSSION There is an increasing requirement for HTP protein engineer- ing methods to meet the demands of drug discovery and the development of chimeric antibodies for human healthcare. In an attempt to meet this demand, the Multisite Gateway tech- nology was reported (7,8). This system assembled multiple DNA sequences and makes domain shufﬂing easily. However, this system required the recombination sequences, an att site, between assembled DNA segments and added several amino acid residues between domains. To improve this point, we developed an HTP N- and C-terminal cloning method using the Gateway system. The Gateway system uses in vitro site- speciﬁc recombination generating clones without requirement for restriction enzyme digestion and ligation. BP reactions are highly efﬁcient and we obtained desired clones using <10 pg PCR fragments for 1 kb targets (data not shown). HTP methods for cloning should allow the handling of numerous samples and generate the desired clones in a short period. We demonstrated that our N- and C-terminal cloning system showed not only reliability, ease of production and screening, but also ability to utilize a wide range of substrates, and that high success rate was shown when 96 samples were handled. Our N- and C-terminal cloning method contained only three steps: PCR, BP in vitro recombination and transfor- mation. The simplicity of the PCR process (mixing primer set, template DNA, reaction solution and enzyme) facilitates establishment of this HTP method. The BP reaction requires simple mixing of PCR products, pDONR vector and reaction mixture without any puriﬁcation step. The transformation step also requires only mixing of samples after the BP reaction and competent cells without any puriﬁcation step. Therefore, all steps can easily be automated. We usually use 50–100 ng template plasmid DNA for 15 cycles PCR with Pfx polymerase (Invitrogen Corp.) and obtained 10–100 ng PCR fragments. In the case of our test gene in this paper, which was 1.5 kb, we obtained the desired clones efﬁciently with 3 ng to 1.6 mg PCR fragments. Fifteen cycles of PCR with high ﬁdelity PCR enzyme, such as Pfx or KOD, gave 1/10 kb error rate from our analysis of more than 10 000 clones. The error rate of synthetic oligo DNA for cloning primer (synthesized by Invitrogen Corp., Japan) was 1/2000 bp according to the analysis of 2500 clones. The test experiment for the HTP method described in this paper failed to gain colonies against ﬁve templates. We characterized 364 clones, 91 target genes · 4 sub-clones, and observed 42 incor- rect clones. This showed that accuracy was 88.5%. Only 2 out