Genomics. 1996 Nov 1;37(3):327-36.

High-efficiency full-length cDNA cloning by biotinylated CAP trapper.

Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, Itoh M, Kamiya M, Shibata K, Sasaki N, Izawa M, Muramatsu M, Hayashizaki Y, Schneider C.

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Genome Science Laboratory, Tsukuba Life Science Center, Ibaraki, Japan.

Abstract

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

PMID:: 8938445; [PubMed - indexed for MEDLINE]

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