) There are many efficient procedures for PCR amplification of representative cDNA populations from different cell types and tissue samples, for the generation of normalized (reduced redundancy) cDNA libraries, and for differential cloning of cDNAs for genes that are preferentially expressed in specific cells or tissues. Almost all of these methods, however, work reliably only with relatively short (@l kb) cDNA fragments but not with full-length cDNAs. In addition, it may be impossible to synthesize full-length cDNA from some tumor RNA preparations, due to RNA degradation in clinical samples. The present application proposes to obviate these problems by developing a cDNA reconstruction procedure that can be used to generate full-length cDNA libraries from high-complexity populations of randomly fragmented cDNA. cDNA reconstruction will be be achieved through multiple steps of cDNA fragment hybridization, heteroduplex extension and PCR amplification of the extended fragments. The reconstruction procedure will be readily combinable with the existing methods for cDNA normalization and subtraction. As the first step in the development of this technique, materials and conditions will be established for the reconstruction of a functional cDNA from two overlapping fragments, at the DNA concentrations corresponding to low-abundance species of cDNA. At the next stage, a procedure will be developed for multistep reconstruction of full-length cDNA starting from <1 kb long cDNA fragments. The reconstructed cDNA clones will be tested for the functionality and sequence conservation and for the extent of recombination between related genes. Subsequently, the procedure will be modified to allow for normalization of the cDNA population during the reconstruction. Once the cDNA reconstruction technique is developed, it will be used to generate and characterize a representational reconstructed cDNA library from a prostate cancer cell line.
| Primiano, Thomas; Baig, Mirza; Maliyekkel, Anil et al. (2003) Identification of potential anticancer drug targets through the selection of growth-inhibitory genetic suppressor elements. Cancer Cell 4:41-53 |
