International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

D11. Cloning Full-Length, Cap-Trapper -Selected cDNAs by Using the Single-Strand Linker Ligation Method

Yuko Shibata1, Piero Carninci1, 2, Akira Watahiki1, 3, Toshiyuki Shiraki1, Hideaki Konno1, 2, Masami Muramatsu1, 2 Yoshihide Hayashizaki1, 2, 3
1Genome Exploration Res Grp, GSC/Genome Science Lab, Tsukuba, RIKEN,
Med, Tsukuba Univ

We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a double-stranded DNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating production of cDNA libraries with titers exceeding 1 x 106 independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' UTRs. The great advantage of our method is that elimination of the GC-tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.

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