79 Article doi:10.1006/geno.2001.6601, available online at http://www.idealibrary.com on IDEAL INTRODUCTION Full-length cDNAs are the starting material for the construc- tion of the RIKEN full-length cDNA encyclopedia. Cloning of full-length cDNA inserts has been hampered by problems related to both the preparation and cloning of long cDNAs. Part of the difficulty associated with the preparation of long cDNAs was overcome with the introduction of a thermosta- bilized and thermoactivated reverse transcriptase [1] and the development of cap-based full-length cDNA selecting tech- niques [2–5]. In contrast to standard cloning techniques, full- length cDNA cloning has the inherent risk of the under-rep- resentation or absence of clones corresponding to long mRNAs in the libraries, because the truncated cDNAs are usually not cloned. However, even in a perfect full-length cDNA library, cDNAs deriving from very long mRNAs will not be cloned if the capacity of the vector is insufficient. Available plasmid cloning vectors show bias for short cDNAs: shorter fragments are cloned more efficiently than are longer ones during the intermolecular competition that occurs at the ligation and library amplification steps. Even though plas- mid electroporation does not show noteworthy size bias, dur- ing circularization of plasmid molecules in the ligation step of a mixed ligation reaction, short cDNAs are ligated more efficiently than are longer cDNAs [6]. In comparison, cloning in -phage vectors is based on the formation of linear concatamers, thus overcoming the prob- lem of size bias. -Phage vectors accommodate cDNAs in a broad range of sizes as well as the high-efficiency cloning of long fragments [7]. Our gene discovery project is based on large-scale sequencing of full-length cDNA libraries. The final product for large-scale sequencing should be a plasmid to facilitate large-scale colony picking, propagation, DNA prepa- ration, and sequencing reactions [8]. A number of -vectors support whole-library bulk excision, but they are suboptimal in terms of cloning size or the excision protocol. Balanced-Size and Long-Size Cloning of Full-Length, Cap-Trapped cDNAs into Vectors of the Novel -FLC Family Allows Enhanced Gene Discovery Rate and Functional Analysis Piero Carninci, 1,2, * Yuko Shibata, 1,2 Norihito Hayatsu, 1,2 Masayoshi Itoh, 1,2 Toshiyuki Shiraki, 1,2 Tomoko Hirozane, 1,2 Akira Watahiki, 1,3 Kazuhiro Shibata, 1,2 Hideaki Konno, 2 Masami Muramatsu, 1,2 and Yoshihide Hayashizaki 1,2,3 1 Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 2 Genome Science Laboratory, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan 3 Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Ibaraki 305-8577, Japan *To whom correspondence and reprint requests should be addressed. Fax: 81-298-36-9098. E-mail: rgscerg@gsc.riken.go.jp. We have developed a new class of cloning vectors: -full-length cDNA (-FLC) cloning vec- tors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, -FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain “size balancers.” Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre- lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The aver- age insert size of the full-length cDNA libraries was correlated to the rate of new gene dis- covery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors. Key Words: full-length cDNA, cloning vector, function vector, normalization and subtraction, gene discovery GENOMICS Vol. 77, Numbers 1–2, September 2001 Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0888-7543/01 $35.00