Balanced-Size and Long-Size Cloning of Full-Length, Cap-Trapped cDNAs into Vectors of the Novel λ-FLC Family Allows Enhanced Gene Discovery Rate and Functional Analysis

• Published in: Genomics (San Diego, Calif.) Calif.), 2001-09, Vol.77 (1-2), p.79-90
• Main Authors: Carninci, Piero, Shibata, Yuko, Hayatsu, Norihito, Itoh, Masayoshi, Shiraki, Toshiyuki, Hirozane, Tomoko, Watahiki, Akira, Shibata, Kazuhiro, Konno, Hideaki, Muramatsu, Masami, Hayashizaki, Yoshihide
• Format: Article
• Language: English
• Subjects: Animals; Bacteriophage lambda - genetics; Base Sequence; Biological and medical sciences; Cloning, Molecular - methods; DNA, Complementary - genetics; Fundamental and applied biological sciences. Psychology; Gene Library; Genes. Genome; Genetic Vectors - genetics; Humans; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Phage ^l; Terminology as Topic
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1006/geno.2001.6601

We have developed a new class of cloning vectors: λ-full-length cDNA (λ-FLC) cloning vectors. 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, λ-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 average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, 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.