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.
□ Subtraction and normalization
In order to efficiently search for cDNA which is present in only trace amounts, it is necessary to reduce the share of high-incidence cDNA occupying the library. For this purpose, DNAFORM offers normalization, to equalize the frequency of various cDNA in the library, and subtraction, which removes non-target cDNA from the library.
Subtraction and normalization takes advantage of the dynamics of hybridization of a single strand nucleic acid with the complementary strand. Namely, we use the phenomenon of higher concentration nucleic acids hybridizing earlier than lower concentration nucleic acids. The remaining mRNA left over from creating cDNAs is biotinylated (the driver) and mixed with an equivalent amount of single-stranded cDNA (the tester: single stranded DNA with a sequence complementary to the mRNA). Under appropriate conditions, the tester and driver are hybridized. cDNA which has hybridized with mRNA under this condition (controlled such that only high-frequency and intermediate-frequency species hybridize), are then selectively removed with the magnetic beads.
□ References
□About the Technology
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P. Carninci et al, High-efficiency full-length cDNA cloning by biotinylated CAP trapper, Genomics, 37, 327?336 (1996)
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P. Carninci et al. High efficiency selection of full-length cDNA by improved biotinylated cap trapper, DNA Res, 28, 61-66 (1997)
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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)
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P. Carninci et al, High-efficiency full-length cDNA cloning, Methods Enzymol, 303, 19-44 (1999)
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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)
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Y. Shibata et al, Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method, Biotechniques, 30, 1250-1254 (2001)
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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)
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Y. Shibata et al, Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing, Biotechniques, 31, 1042-1044 (2001)
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P. Carninci et al, Extra-long first-strand cDNA synthesis, Biotechniques, 32, 984-985 (2002)
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Y. Shibata et al, Cytoplasmic RNA extraction from fresh and frozen mammalian tissue, Biotechniques, 33, 306-309 (2002)
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P. Carninci et al, Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia, Genome Res, 13, 1273-1289 (2003)
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T. Hirozane-Kishikawa et al, Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts, Biotechniques , 35, 510-518 (2003)