Screening of lambda library for differentially expressed genes using in vitro transcripts.

Abstract

We present an improved approach to the screening of eucaryotic libraries for differential gene expression. Previous techniques have generated probe via the harvesting of cellular poly(A)+ RNA and synthesizing labeled cDNA probe using reverse transcriptase. In our approach we prepare labeled RNA probe via in vitro transcription. Unlike cDNA preparation, in vitro transcription (i) directly reflects the ongoing rate of gene expression, and (ii) allows one to assess expression of genes whose transcripts are not polyadenylated. To make this approach practical for the screening of a large library, we modified and optimized existing in vitro transcription techniques, enhancing manyfold the [alpha-32P]UTP incorporation into mRNA, while almost completely suppressing rRNA incorporation. In addition, we developed a simple procedure for making precise replicate dot blots of very large quantities of lambda-phage library DNA. By combining our techniques of in vitro transcription and replicate blotting, we are able to approach detection of a twofold difference in gene expression over a greater than 1000-fold range in overall expression. Our single-clone amplification and dot-blotting technique resulted in nearly the same number of lambda-phage DNA copies per dot for all members of the library. This feature allows us to assign genes to different expression classes, as well as to detect any alterations in expression. We demonstrate our approach by screening the drosophila genomic DNA library with in vitro transcripts from drosophila tissue culture cells. Screening of the entire drosophila genomic library at the 99% probability level is readily achieved.