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.

Cap-Trapper™ method technology overview

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.


About the Technology