Reusable cDNA Libraries for cDNA Amplification and Cloning

Solid-Phase cDNA synthesis

When preparing solid-phase cDNA libraries, you first capture the mRNA onto the Dynabeads. The beads are then added directly to the RT-reaction, where the bead-bound oligo-dT residues act as primers for first strand cDNA synthesis. As all operations utilize the solid-phase, buffer changes are fast, simple and effective.

Re-Usable cDNA library

A stable, covalently linked cDNA library is produced and can be used over and over again (e.g for PCR-amplification, RACE, subtractive hybridisation, cDNA cloning etc.). Optimal buffer conditions are achieved by simply magnetic separation and resuspension in the appropriate buffer. Reverse transcription will only be limited by the efficiency of the enzyme used.

The bead-bound probe used both for capture and primer can be oligo-dT or any specific DNA sequence. The patented Dynabeads solid-phase cDNA synthesis technology is compatible with a various commercially available cDNA kits.

The use of Dynabeads Oligo(dT)₂₅ for capture of mRNA for cDNA synthesis and/or subtractive cloning is covered by the patent "Process for producing" cDNA (WO9006044, EPO444119, AT122721T, DE68922743T, DE68922743D, CA2003500, AU627815, US5759820 and JP2960776B2).

Selected References

1. Jakobsen KS et al. (1994). Direct mRNA Isolation Using Magnetic Oligo (dT) Beads: A Protocol for All Types of Cell Cultures, Animal and Plant Tissues. Advances in biomagnetic separation. Eaton Publishing 61-71.
2. Karrer EE et al. (1995).In situ isolation of mRNA from individual plant cells: creation of cell specific libraries. Proc.Natl.Acad.Sci.USA 92:3814-3818.
3. Lambert KN and Williamson VM (1993). cDNA library construction from small amounts of RNA using paramagnetic beads and PCR. Nucleic Acids Research 21(3):775-776.
4. Raineri I, Moroni C and Senn HP (1991). Improved efficiency for single-sided PCR by creating a reusable pool of first-strand cDNA coupled to a solid phase. Nucleic Acids Res. 19(14):4010.
5. Fellmann F, Pretet J and Fellmann D (1996). Simplified Protocol of Solid-Phase cDNA libraries for Multiple PCR Amplification. BioTechniques 21(5): 766-770.
6. Rodriguez IR et al.(1994). Structural Analysis of the Human Hydroxyindole-O-methyltransferase Gene. J.Biol.Chem. 269(50):31969-31977.
7. Aasheim HC, Logtenberg T and Larsen F. (1996). Subtractive Hybridization for the Isolation of Differentially Expressed Genes Using Magnetic Beads. Methods in Molecular Biology 69:115-128.