Construction and characterization of a full length-enriched and a 5′-end-enriched cDNA library
Introduction
Messenger RNA of eukaryotes has a cap structure at its 5′ end (Furuichi and Miura, 1975) and a poly(A) stretch at its 3′ end (Darnell et al., 1971; Edmonds et al., 1971; Lee et al., 1971). The sequence information between the cap and the poly(A) is important for identification of the coding region and the non-coding region that control mRNA stability and translation efficiency. Thus, isolation of a `full-length' cDNA, which consists of a copy of all the sequences between the cap and the poly(A) of a mRNA, is an indispensable step for the analysis of gene structure and function.
The conventional cDNA library has several drawbacks for isolating `full-length' cDNAs. One is its low content of cDNA clones whose 5′ ends extend to their mRNA start sites. Another more serious flaw is the difficulty in identifying the cDNA clones that contain `full-length' cDNA. In the case of the 3′ end, the identification is simple because poly(A) can serve as the sequence tag for the 3′ end. In contrast, the cap structure cannot be the tag for the mRNA start site.
Previously, we and others have developed a `oligo-capping' method that allows us to replace the cap structure with synthetic oligonucleotides (Fromont-Racine et al., 1993, Maruyama and Sugano, 1994). The synthetic oligonucleotides used to replace the cap structure (5′-oligo) can serve as the sequence tag for the mRNA start site. If we use `oligo-capped' mRNAs and oligo-dT primer for the construction of a cDNA library, we can identify the `full-length' clones easily by monitoring their 5′ and 3′ end sequences. We can also selectively clone `full-length' cDNAs into a vector, if appropriate restriction sites are introduced into the 5′-oligo and oligo-dT primers. Furthermore, one can clone the 5′ ends of mRNAs selectively by a similar procedure using random primers for cDNA synthesis. This type of 5′-end-enriched cDNA library may be useful for the isolation of the 5′ ends of mRNAs, which would be difficult to isolate from an oligo-dT primed cDNA library. In this report, we will describe the construction and the characterization of these two types of cDNA library.
Section snippets
Cell
Human neuroblastoma cell line SK-N-MC was obtained from the American Type Culture Collection and grown as described by Biedler et al. (1973).
Isolation of RNA
Cytoplasmic RNA and poly(A)+ RNA were isolated according to the standard method (Sambrook et al., 1989). Oligo-dT cellulose was from Collaborative Biomedical Products.
Vector
pUC19-fl2 is constructed by cloning the 453-bp EcoRI–XbaI fragment of pME18 (Xie et al., 1995) into the EcoRI and XbaI site of pUC19. After cloning, one DraIII site containing
Construction of a full length-enriched and a 5′-end-enriched library
According to the scheme shown in Fig. 1, we constructed a full length-enriched and a 5′-end-enriched cDNA library using poly(A)+ RNA from human neuroblastoma cell line SK-N-MC. Details of the procedures are described in the Materials and methods. The size of the cDNA library was about 20 000 clones/μg of poly(A)+ RNA for the full length-enriched cDNA library and about 200 000 clones/μg for the 5′-end-enriched cDNA library. The average length of cDNA inserts of 20 cDNA clones from each library
Conclusion
In this report, we described the construction of new types of cDNA library. One is a full length-enriched cDNA library, and the other is a 5′-end-enriched cDNA library. The content of cDNA clones that have the mRNA start site in both libraries was estimated at around 80%. This is a significant improvement of the content compared to the cDNA libraries made by conventional methods. Furthermore, the 5′-end-enriched cDNA library seems to contain the cDNA clone with the mRNA start site of the long
Acknowledgements
We would like to dedicate this work to the late Professor Nobuo Yamaguchi who died in an accident on 27 January 1996. He generously supported this work and constantly gave us encouragement. We would also like to thank Miss Naoko Wakairo for her excellent sequencing work. This work was supported by Grants-in-Aid for Creative Basic Research (Human Genome Program) and Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture.
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