The development of functional genomics resources is essential to understand and utilize information generated from genome sequencing projects. Central to the development of this technology is the creation of high quality cDNA resources and improved technologies for analyzing coding and noncoding mRNA sequences. In fact, the characterization of the full coding potential of a particular mRNA is the essential starting point when undertaking to investigate biological function. A full length cDNA allows one to predict transcription and translation initiation start sites, deduce certain protein characteristics based on primary amino acid sequence, predict transcription termination sites, and visually inspect the 5' and 3' untranslated regions for elements which may be involved in post- transcriptional regulation of gene expression. The analysis of several complete cDNAs for a given gene enables one to gather information on alternative splicing, alternative promoter usage, and alternative polyadenylation signals all events known to be important in regulating gene expression. The comparison of genomic and cDNA sequence is necessary for determining exon-intron structure and to document the occurrence of RNA editing - a post-transcriptional regulatory mechanism on which we have little information. In the current proposal, we aim to continue developing technologies for generating full-length cDNAs, and incorporate such technology into a process to yield high quality full-length cDNA libraries.
Our specific aims are: i) To improve the efficiency and quality of first strand synthesis, ii) Establish a method that will determine whether a given set of cDNAs are full length, iii) Generate 5' selected cDNA libraries employing an improved affinity method, and iv) Implement an approach utilizing homologous recombination in E. coli by which the structure of cDNA clones can be tailored to individual needs. Together, these studies should yield higher quality cDNA libraries that can provide an important resource for novel gene discovery and provide reagents necessary for functional studies.
| Das, M; Burge, C B; Park, E et al. (2001) Assessment of the total number of human transcription units. Genomics 77:71-8 |
| Das, M; Harvey, I; Chu, L L et al. (2001) Full-length cDNAs: more than just reaching the ends. Physiol Genomics 6:57-80 |
