e109 Nucleic Acids Research, 2005, Vol. 33, No. 12 PAGE 4 OF 6 Table 2. Relation between the length of homologous region and cloning efficiency for the target containing tandem repeats NH1000 · HC474 NH1022 · HC474 NH1000 · HC816 NH1000 · HC836 NH1022 · HC816 Combination of fragments End homologous region (bp) Number of colonies Recombination between the end of N-terminal and C-terminal fragments Recombination between the end of N-terminal fragment and inside ‘rep seq’ of C-terminal fragment Others 0 132 – 12/16 4/16 0 37 – 8/16 8/16 26 124 6/16 5/16 5/16 46 76 15/16 1/16 0/16 48 195 14/16 1/16 1/16 Table 3. Relation between the amount of N- and C-terminal fragments and cloning efficiency Ratio between 1/32 1/16 1/8 1/4 1/2 1/1 2 vector and fragment Number of 15 14 58 177 203 362 425 397 273 157 77 colonies Rate of right 1/8 6/8 6/8 5/8 7/8 6/8 8/8 8/8 8/8 7/8 7/8 clone 4 8 16 32 obtained by the end recombination was 6/16. The ratio of the recombination between ‘rep seq’ of the N-terminal fragment and the mid C-terminal fragment was 5/16. The ratio of the recombination among other homologous sequence was also 5/16. The combinations HN1000/HC836 and HN1022/ HC816 showed 46 and 48 bp homologous region at the ends, respectively, and the frequency of the end recombination of these combinations was very high at 15/16 and 14/16, respect- ively. These results suggested that the combination of N- and C-terminal fragments with long, ( 40–50 bp), homologous regions at their ends produced the desired clones with high reliability even if the gene had short homologous regions, such as 30 bp identical sequence. The amount of N- and C-terminal fragments required for cloning We analyzed the range and suitable amount of DNA fragments for cloning. We selected the combination N959/C709, whose length of end homologous region was 39 bp, for this analysis. We measured colony forming ability and reliability of cloning with a combination of different molar ratios of fragments and 150 ng pDONR vector, from 1/32:1/32:1 to 32:32:1. The amount of PCR fragment was from 1.56 to 1636 ng. After the BP reaction, we introduced linear plasmid, which had 37.5 ng vector DNA, into E.coli and counted the number of colonies. We selected eight colonies from each experiment and sequence analyzed these clones. The results are summar- ized in Table 3. For cloning of this 1.5 kb gene, the optimal ratio of fragments and a vector was 2:1. Using this condition, 425 colonies were obtained with all eight sequenced clones having the desired sequence. This experiment showed that the desired clone was obtained with high reliability using a wide variation in the relative quantity. Even when using <2 ng of insert fragment, 3% of vector DNA gave the right clone although the rate of the right clone was only 1/8. HTP Gateway cloning with N- and C-terminal fragments HTP methods require simple procedures, which will work for a wide range of amount of substrates to reliably complete each reaction. The results of all experiments described above showed that the N- and C-terminal cloning method using the Gateway system incorporate not only easy handling proced- ures and high cloning efﬁciency but also that the reliability of this method was very high when the ends of N- and C-terminal fragments were designed to carry 40 bp or longer homologous sequences for recombination in E.coli. These results demon- strate that our N- and C-terminal cloning method was suitable for HTP cloning and chimeragenesis. We measured the success rate of this method for HTP handling with 96 target genes. These samples were randomly chosen from human full-length cDNA library. The sizes of their open reading fragment were from 603 to 3381 bp. Among these clones, 37 clones had 603–1000 bp, 39 clones had 1001– 2000 bp and 20 clones had more than 2000 bp long target regions for cloning. The primers for ampliﬁcation of N- and C-terminal fragments were designed to produce 30–60% full- length fragments containing 40 bp end homologous sequences. Ninety-six combinations of N- and C-terminal fragments were mixed with 150 ng pDNOR201 vector and in vitro BP recomb- ination reactions were carried out. One-fourth of each reaction mixture was used to transform competent E.coli and the cells were plated on LB containing 50 mg/ml of Kanamycin. After the colonies were counted, four colonies from each plate were randomly selected, and the length of inserts and their sequences were analyzed. The ampliﬁed N- and C-terminal fragments and inserts of clones are shown in Figure 4a. Cloning efﬁciency and rates of the desired clones are illus- trated in Figure 4b and c, respectively. No colonies were obtained from ﬁve targets, four of which did not produce N- or C-terminal fragments by PCR (indicated by green dots in Figure 4a). A total of 66 out of 96 reactions (69%) gave only correct clones and 16 out of 96 (17%) gave only one wrong clones when four clones from each point were analyzed. More than 85% (82 out of 96) gave the desired clones with high reliability. Cloning failed completely for only 7 out of 96 targets using this system equating to a 93% success rate when four clones from each reaction were checked. Introduction of mutations The N- and C-terminal Gateway cloning method was also applicable to site-directed mutagenesis, introducing point mutation, deletion, insertion or substitution at any position in the gene according to the primer design. When two priming