e109 Nucleic Acids Research, 2005, Vol. 33, No. 12 of 42 incorrect clones showed a base substitution error. The other showed different sizes of inserts or vector background. Such size differences were easily observed by colony PCR and gel electrophoresis. This suggested that the accuracy of this HTP method was very high and colony PCR was sufﬁcient for selection of desired clones. All of our results demonstrated that the N- and C-terminal cloning method described here was suitable for HTP chimeragenesis and site-speciﬁc mutagen- esis. Since two types of pDONR vector carrying different anti- biotics resistant genes, Kanamycin and Zeocin, exist, serial N- and C-terminal cloning for chimeragenesis and domain shufﬂing are possible. The length of insertion and substitution depends on the length of reliable oligo primers. Reasonable length for the reliable primers is 80mer. This length creates an 80 bp insertion because 40 bp is required for homologous recombination in E.coli and 20 bp is used as the priming site for PCR. We established one-step adapter PCR, using four primers in one tube PCR, as described previously (9). This method allowed a 200 bp insertion. Serial cloning also gives longer insertion and substitution. Translocation of chromosomes produces chimeric proteins underlying many diseases, including hematological malignan- cies and solid tumors. Mitelman et al. (10) reported that 209 fusion genes on hematological malignancies and 271 fusion genes on solid tumors. The characterization of chimeric proteins is important to understand the mechanism of such diseases. However, one of the biggest problems is the difﬁ- culty to obtain full-length cDNA clones with fusion from patents. Recently, several projects produced huge number of human full-length cDNAs (11,12), which covered most genes of human. The method presented here can create all chimeric clones using the established full-length cDNAs and will open the cyclopedic study on functional analyses of fusion proteins in cancer. In this paper, we introduced the method for protein engin- eering included chimera protein production. Our method was for assembling two DNA fragments and constructing plasmids by HTP way. This method allowed introducing any kinds of sequences, such as regulatory sequences, localization signals and control elements at any place of genes and had the wide application. We believe that this method helps the works of many research ﬁelds. PAGE 6 OF 6 ACKNOWLEDGEMENTS The authors thank Drs J. Chesnut and M. Jones for critical reading of the manuscript. This work was supported by a grant from the NEDO Project of Ministry of Economy, Trade and Industry. Funding to pay the Open Access publication charges for this article was provided by Invitrogen Japan, Invitrogen Corporation. Conflict of interest statement. 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