Edition 4- November 1997

cDNA libraries: now also available from BCCM™/LMBP

A cDNA library is an array of DNA copies of a mRNA population that are propagated in a cloning vector (plasmid or phage) and often maintained in Escherichia coli.

A good cDNA library is large enough to contain full-length representatives of all sequences of interest, some of which may be derived from low-abundance mRNAs. A typical mammalian cell contains 10,000-30,000 different mRNA sequences. To reach a 99% probability of including a low copy sequence, a cDNA library consisting of 1.7x10⁵ clones would have to be screened. However, very rare messages can be as low as one in 10⁶.

Depending on the research objective, cDNA libraries can be either unidirectional or bi-directional. In the former class the cDNA inserts are cloned in a specific orientation relative to the transcriptional polarity of the original mRNAs, whereas the latter class contains cDNA inserts cloned in either orientation. Expression libraries constitute a specialised form of unidirectional insertion and are particularly useful, for instance, when screening for a desired gene is based on biological function or when antibodies are used to isolate the target gene.

The cDNA libraries offered by BCCM™/LMBP are prepared using a protocol based on the Gubler-Hoffman Procedure. SfiI-NotI cDNA is obtained by using a oligo(dT)-NotI primer-adapter in the first strand synthesis, ligating a SfiI adapter to the blunted cDNA, and digesting the cDNA with the NotI restriction enzyme (Goldman et al, 1992). The cDNA is ligated in the cloning vector and electroporated in E. coli. All cloning vectors used in our cDNA libraries are plasmid vectors.

The quality of the library is checked by determining the insert sizes in a representative number of recombinants digested with the cloning enzymes. The average size is approximately 1200 bp. Using PCR with primers of a housekeeping gene, or comparative hybridisation with oligonucleotide probes from the beginning and the end of the housekeeping gene (e.g. actin), we demonstrate that, on average, 50% of the cDNA clones are full-size. At present BCCM™/LMBP offers some 14 high quality libraries. Salient features of some representative libraries are given in the table below.

References:
Goldman, G.H., J. Demolder, S. Dewaele, A. Herrera-Estrella, R. Geremia, M. Van Montagu and R. Contreras, Mol. Gen. Genet. 234, 481-488 (1992)

Contact
Prof. R. Contreras,
BCCM™/LMBP
Tel. +32 9 264 51 36
Fax. +32 9 264 53 48
E-mail: roland.contreras@lmb.rug.ac.be

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