Genetic screening - For the experts

By means of the PCR technique we are able find related, but yet different gene products. This technique can improve the knowledge of the diversity of enzymes and thereby improving the productivity of enzymes under different conditions.

At Novozymes we discover new enzyme genes using molecular screening and screening using DNA microarrays. Both methods eliminate the need for a functional assay to detect the gene.

Cloning of new enzyme genes often relies on expression of the gene in question or characterization of the gene product. For example, cloning of cDNA requires the presence of the corresponding mRNA. Screening of an expression library necessitates an assay to detect the enzyme activity. A probe used for screening a genomic library is designed from information about the corresponding protein sequence.

Molecular screening uses the information in sequences from a set of related enzyme genes to clone additional genes from other organisms. An alignment of either the nucleotide sequences or the corresponding aminoacid sequences followed by identification of evolutionarily conserved regions makes it possible to design degenerate PCR primers containing the DNA sequence corresponding to the homologous region. These primers can be used to amplify and sequence a fragment of a homologous gene from a sample of genomic DNA. Using this method, a number of genes homologous to the initial gene sequences can relatively quickly be identified.

Using this method, we succeeded very rapidly in cloning 32 cellulase genes representing all 4 major fungal taxa: Ascomycota, Zygomycota, Chytridiomycota and Basidiomycota based on 4 homologous gene sequences known at the start of the project.