Several attempts have been made to make cDNA libraries from small amounts of tissues utilizing PCR to amplify the cDNA. These cDNA libraries were inadequate for several reasons. We propose to develop new techniques to prepare high quality cDNA libraries from small numbers of functionally defined cells obtained by microdissection. In Phase I, lambda cDNA libraries will be synthesized from ovarian follicles dissected from frozen sections. Several improved techniques have been incorporated into the cDNA synthesis and cloning methodology. The new techniques include the use of a novel oligo (dT) cellulose matrix, which will allow the use of PCR primers without homopolymeric sequences. Furthermore, the use of Pfu DNA polymerase for amplification will result in cDNAs with fewer mutations than with Tag DNA polymerase. Techniques are described for quality control of the cDNA products at each step of the process. The criteria for cDNA library quality are also defined. The development of these techniques will permit cDNA library construction from small reproductive and non-reproductive structural-functional regions of adult and embryonic tissues. It will also lay ground work for the preparation of cDNA libraries from single cells (e.g., male germ cells at various stages of spermatogenesis), which will allow the investigation of cell types in their native environment.
Commercially, tissue region-specific cDNA libraries will replace whole tissue libraries, which are now marketed by several companies. Region- specific libraries will better represent functionally-defined cell types. DNA libraries can also be custom made for investigators. Also, the amplified cDNAs are amenable to the preparation of custom subtractive and normalized cDNA libraries.
