Construction of cDNA libralies by oligo-capping method

A full-length cDNA can be an important material for empirical analysis of the function of gene products. However, it has been extremely difficult to efficiently isolate full-length cDNAs from the cDNA libraries constructed by conventional methods. In these methods, mRNAs extracted from biological samples have a high content of incomplete-length mRNAs which have been degraded, and in many cases cDNAs originating from these incomplete mRNA molecules are cloned. The method established by Gubler and Hoffman (1) is widely used because it facilitates the isolation of relatively long cDNAs. However, this method theoretically does not allow the synthesis of cDNAs corresponding to the 5' terminal regions of mRNAs because the mRNA annealed to the first strand is cut to be used as a primer for the second-strand cDNA synthesis.

Sugano et al. developed the "oligo-capping method" (2-3) which replaces the cap structure specific to the 5' end of eukaryotic mRNA with a synthetic oligonucleotide (Fig. 3-1, Fig. 3-2 & Fig.3-3). Oligo-capping method consists of: i) removing phosphate from the 5' end by treating mRNA samples with bacterial alkaline phosphatase (BAP); ii) converting the cap structure into a phosphate by treatment with tobacco acid pyrophosphatase (TAP). This treatment results in full-length mRNAs which initially had the CAP structure as the only mRNAs with phosphate at the 5' end; iii) a synthetic oligo RNA "oligo-cap linker" is ligated specifically to the 5' end of the full-length mRNA using RNA ligase. This method enables us to isolate full-length cDNAs efficiently by constructing cDNA libraries from these mRNAs with a specific sequence ligated to the 5' end. Using the mRNA with the attached oligo-cap linker as a template, first-strand cDNAs are synthesized. This is followed by PCR using as primers the oligo-cap linker sequence and an oligo-dT-adapter. This PCR produces a "oligo-capped, full-length-enriched cDNA library" which has a high content of full-length cDNA corresponding to the region from the 5' end to the 3' end of each of the full-length mRNAs.

We improved the oligo-capping method by optimizing all steps (4). Full-length rates of human cDNA libraries constructed by Helix Research Institute using the improved oligo-capping method were 90% and the majority of the cDNA insert sizes was over 2 kb.

References

(1) Gubler, U. and Hoffman, B. J. (1983) Gene 25:263-269

(2) Maruyama, K. and Sugano, S. (1994) Gene 138: 171-174.

(3) Suzuki, Y. et al. (1997) Gene 200: 149-156.

(4) Ota, T. et al., WO 01/04286