GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP

The release of vast quantities of DNA sequence data by large-scale genome a nd expressed sequence tag (EST) projects underlines the necessity for the d evelopment of efficient and inexpensive ways to link sequence databases wit h temporal and spatial expression profiles, Here we demonstrate the power o f linking cDNA sequence data (including EST sequences) with transcript prof iles revealed by cDNA-AFLP, a highly reproducible differential display meth od based on restriction enzyme digests and selective amplification under hi gh stringency conditions, We have developed a computer program (GenEST) tha t predicts the sizes of virtual transcript-derived fragments (TDFs) of in s ilico-digested cDNA sequences retrieved from databases. The vast majority o f the resulting virtual TDFs could be traced back among the thousands of TD Fs displayed on cDNA-AFLP gels, Sequencing of the corresponding bands excis ed from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles, The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction e nzyme recognition sites, the primer extensions and the estimated TDF size a s identifiers, the DNA sequence(s) corresponding to a TDF with an interesti ng expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostoch iensis, Various novel pathogenicity factors were identified by combining ES Ts from the infective stage juveniles with expression profiles of similar t o 4000 genes in five developmental stages produced by cDNA-AFLP.