PAGE 3 OF 6 homologous sequences when using the N-term C-term cloning method. To address this question we constructed a clone con- taining a tandem repeat. We chose a 1732 bp cDNA for the test gene. This cDNA carried two 26 bp identical sequences, named ‘rep seq’ sites, and several homologous regions, whose size and homology were 10–30 bp and 70–90%. The ‘rep seq’ sites are indicated with blue boxes in Figure 3. We Figure 2. Preparation of N- and C-terminal fragments with different lengths of homologous sequences on the ends. Five N-terminal and one C-terminal frag- ments were prepared by PCR. The combinations of N- and C-terminal frag- ments gave 10, 20, 39, 78 and 133 bp homologous sequences on the ends of each fragment. Table 1. Relation between the length of homologous region and efficiency of cloning Length of homologous region Number of colonies Rate of right clone 10 bp 20 bp 39 bp 78 bp 133 bp 25 6/6 110 6/6 334 6/6 367 6/6 406 6/6 Nucleic Acids Research, 2005, Vol. 33, No. 12 e109 prepared two N-terminal, HN1000 and HN1022, and three C-terminal fragments, HC836, HC816 and HC474, by PCR. HN1000 and HN1022 fragments carried ‘rep seq’ sites at 30 end and 22 bp inside of the end, respectively. Both HC816 and HC836 had two ‘rep seq’ sites, one proximal and one distal. The proximal ‘rep seq’ sites of HC816 and HC836 were loc- ated at 50 end and 22 bp inside of the end, respectively. The distance between the end and distal ‘rep seq’ site was several hundred base pairs. HC474 had only a distal ‘rep seq’ site. We measured the cloning efﬁciency and reliability with various combinations of N and C fragments. The combinations HN1000/HC836, HN1000/HC816 and HN1022/HC816 car- ried 46, 26 and 48 bp homologous sequence at their ends and a 26 bp homologous sequence of ‘rep seq’ sites, respect- ively. The combinations HN1000/HC474 and HN1022/HC474 had no end homologous sequence but a 26 bp homologous sequence of ‘rep seq’ sites. We randomly selected 16 colonies from each experiment for characterization. The number of colonies obtained from 37.5 ng vector DNA and the ratio of correct clones are shown in Table 2. Sequence analysis of the resultant clones from the combinations HN1000/ HC474 and HN1022/HC474 showed that recombination had occurred between ‘rep seq’ sites at the end of the N-terminal fragment and the mid region of the C-terminal fragment. The ratios of homologous recombination between two ‘rep seq’ sites obtained by the combinations HN1000/ HC474 and HN1022/HC474 were 12/16 and 8/16, respect- ively. The remaining clones in these experiments showed recombination between the other homologous regions. The combination HN1000/HC816, which had a 26 bp homologous region at both ends of N- and C-terminal frag- ments, was tested for efﬁciency to produce speciﬁc designed clones by end recombination. The ratio of designed clone Figure 3. Preparation of N- and C-terminal fragments from a template gene carrying identical sequences. The target gene had two identical sequences named ‘rep seq’ indicated by blue boxes. Two N-terminal fragments, HN1000 and HN1022, and three C-terminal fragments, HC836, HC816 and HC474, were prepared by PCR. HC836 and HC816 had two ‘rep seq’ on their 50 ends and inside of the fragments. The other fragments had only one rep seq’.