The cloning of full-length cDNAs is an expensive and time- consuming, but obligatory step, for many molecular genetic studies. The intermediary steps from the first identification of a cDNA clone to elucidation of full-length cDNA sequence can require months of intensive experimentation. Whole-genome strategies including microarray expression analysis, comparative genomic analyses, and re-sequencing projects to identify disease- related sequence variants all rely upon the elucidation of full- length cDNAs. Construction of full-length cDNA libraries has been thwarted in the past by technical limitations, many of which are associated with methods to handle and prepare mRNA. Recent advances point the way to overcome these limitations.
The aim of the proposed research is to construct a set of representative, full-length mouse cDNA libraries of sufficient quality to allow identification of full-length cDNA clones corresponding to the vast majority of genes in a single library screen. Libraries will be constructed to optimize both functional studies and DNA sequencing. To achieve this ambitious goal, we present plans to improve current protocols for tissue collection, mRNA isolation, reverse transcription, and long-range PCR. These protocols will be integrated with novel techniques to enhance selection of full-length cDNAs while simultaneously eliminating degraded or prematurely terminated templates. Pools of cDNA clones will be normalized by an established protocol using three cycles of reannealing followed by PCR amplification to insure equal representation of genes expressed at different levels in different tissues. Lastly, we present a scheme to quantitatively evaluate the size, integrity, and representation of mRNA and cDNA at all critical steps of library preparation.
