International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

Table of Contents * Genes, Physical Mapping and Sequencing * Gene Identification * Bioinformatics * Common Resources * RIKEN Session * Functional Genomics * Complex Traits * Mutagenesis * Developmental Genetics and Differentiation * Verne Chapman Memorial Lecture I and II

D13. Construction of Shotgun Libraries for the Mouse Full-Length cDNA Clones

Masayasu Yoshino , Jun Kawai , Akira Shinagawa , Kazuhiro Shibata , Masayoshi Itoh , Piero Carninci , Yoshihide Hayashizaki
Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC) and Genome Science Laboratory, RIKEN Tsukuba Institute, Koyadai 3-1-1, Tsukuba 305-0074, Japan Core Research of Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST)

To determine the complete sequence data of the mouse full-length cDNA (FL-cDNA) clones, we developed a system for the high-throughput preparation of shotgun libraries for the FL-cDNAs. Eight libraries out of 384 clones are routinely made everyday by nine staffs in our lab. The procedure is summarized as follows:

1) The FL-cDNA clones were sorted according to their insert size, which is required to pool multiple clones into a single shotgun library.

2) The cDNA inserts were amplified by PCR to remove the vector from the clones.

3) The concatenation methodology (Yu et al. 1997, Genome Research 7: 353-358) was adapted to get random fragments even from small cDNAs of < 3-kb.

4) The 1-2 kb randomly-sheared fragments were obtained by use of ouble Stroke Shearing Device (Fiore Automation, Inc.)

5) The DNA-ends were blunted and sub-cloned into a plasmid vector.

In our preliminary data, about 100 reads are required to determine the complete sequence of a 3-kb cDNA insert by use of the RISA sequencer. The sequence data produced by the present shotgun method are included in the report by Kawai et al. in this meeting.