Directional cDNA library construction

For poorly characterized genomes or in circumstances where a particular tissue or developmental stage is not well-represented in the available resource of cDNA libraries, one may wish to array cDNA clones directly from libraries constructed in-house. Ideally, it would be preferable to have a limited duplication of cDNA clones on a microarray. If random clones are chosen from a cDNA library then the more abundantly expressed sequences will dominate the microarray. Therefore a sequence verified nonredundant set of clones is a better option. Dependant on the species of interest, such a set of clones may be available commercially or from academic establishments (such as a sequence verified set of human IMAGE consortium clones available from Research Genetics and soon to be available from the HGMP resource centre in the UK). However it must be noted that all sources of sequence verified clones will have some error in clone identification. Furthermore, novel and previously ill-defined genes will be unrepresented on the array. 

The use of free primers requires that the double-stranded form of the cDNA synthesized be subsequently ligated to a linearized vector by one of the following three methods [see Fig. 6.6(1)]. The first is a direct, low efficiency blunt-end ligation between double-stranded cDNA and a blunt-ended vector. This is the most straightforward method to clone cDNA from abundant mRNA species and is not suitable for cDNA library construction. 

By 1980, investigations on the molecular biology of malaria under the direction of David Kemp had begun. Two PhD students, Ross Coppel and Alan Cowman, were able to construct cDNA libraries with Ross working on P. falciparum and Alan on Babesia bovis. Work on P. falciparum was... 

ISOLATION OF INTACT mRNA AND CONSTRUCTION OF FULL-LENGTH cDNA LIBRARIES USE OF A NEW VECTOR, Agt22, AND PRIMER-ADAPTERS FOR DIRECTIONAL cDNA CLONING... 

Confirmation of the proposed carboxy-terminal sequence was very recently obtained from the nucleotide sequence of a complementary DNA (cDNA) segment coding for the carboxy-terminal portion of RBP (Costanzo et al., 1983). A human liver cDNA library was constructed and a large number of individual clones were screened directly by cDNA sequence analysis. One of the 236 sequences screened coded for the region of human RBP from amino acid residue 159 to 182. 

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