Custom Full-Length cDNA Service : Technology Overview

□ Technical Information

□Cap-Trapper™ method

DNAFORM full-length cDNA libraries are created using the Cap-Trapper™ method, which has been developed at the RIKEN. It allows the creation of full-length cDNA libraries containing a high percentage of full-length cDNA (up to 95%), starting from a small amount of total RNA.

In the Cap-Trapper™ method, the cap structure unique to eukaryotic mRNA is first biotinylated. The cap structure is then selectively removed from partial cDNA fragments by processing with RNase I. Remaining full-length cDNA, which still has an intact cap structure, is biotinylated with strepatvitin coated magnetic beads and captured. This selected full-length cDNA is eluted from the beads with alkaline treatment. Finally, by synthesizing the second strand from the initial single strand cDNA eluted from the beads, one can selectively obtain the full-length cDNA.

□ References

□About the Technology

P. Carninci et al, High-efficiency full-length cDNA cloning by biotinylated CAP trapper, Genomics, 37, 327?336 (1996)
P. Carninci et al. High efficiency selection of full-length cDNA by improved biotinylated cap trapper, DNA Res, 28, 61-66 (1997)
P. Carninci et al, Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA, PNAS 25, 520-524 (1998)
P. Carninci et al, High-efficiency full-length cDNA cloning, Methods Enzymol, 303, 19-44 (1999)
P. Carninci et al, E. Valen et al, Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes, Genome Res, 10, 1617-1630 (2000)
Y. Shibata et al, Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method, Biotechniques, 30, 1250-1254 (2001)
P. Carninci et al, Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis, Genomics, 77, 79-90 (2001)
Y. Shibata et al, Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing, Biotechniques, 31, 1042-1044 (2001)
P. Carninci et al, Extra-long first-strand cDNA synthesis, Biotechniques, 32, 984-985 (2002)
Y. Shibata et al, Cytoplasmic RNA extraction from fresh and frozen mammalian tissue, Biotechniques, 33, 306-309 (2002)
P. Carninci et al, Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia, Genome Res, 13, 1273-1289 (2003)
T. Hirozane-Kishikawa et al, Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts, Biotechniques , 35, 510-518 (2003)