Construction of cDNA Library

Ligate with vector that has been cut with EcoR1 

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Fietto, J.L., DeMarco, R. and Verjovski-Almeida, S. (2002) Use of degenerate primers and touchdown PCR for construction of cDNA libraries. Biotechniques 32, 1404-1411. 

Brady, G., and Iscove, N. N. (1993) Construction of cDNA libraries from single cells. Methods Enzymol. 225, 611-623. 

The reverse genetic approach relies on the construction of suitable libraries. The initial step is to homogenize the tissue and isolate the mRNA population. As mRNAs are single stranded and relatively unstable, they are first converted to their complimentary DNA (cDNA) sequence, and then into the more stable double-stranded cDNA molecule. The next step is to find a suitable vector that can carry the cDNA molecule and replicate itself multiple times. This will allow each message to be amplified to the large numbers of copies required for isolation and purification. 

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. 

Li, Y., Yang, L., Cui, J.T., Li, W.M., Guo, R.F., and Lu, Y.Y. 2002c. Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells. World J Gastroenterol 8(2) 208-212. 

Jiang, G. and Harrison, D. J. 2000. roRNA isolation in a microfluidic device for eventual integration of cDNA library construction. Analyst 125 2176-2179. 

A.aculeatus CBS 115.80 was grown on apple pectin in a fermenter and strong induction of Rgase production could be observed. Total RNA was isolated from such an RGase producing culture and used for the construction of a cDNA library. 

A very similar cloning strategy has been used for the 48-kDa subunit of the glycine-receptor channel by Betz and collaborators [4]. Strychnine was used as an affinity ligand, and synthetic oligopeptides were constructed from the purified material. They have been used to screen cDNA libraries. 

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