Plant & Animal Genome IX Conference Plant & Animal Genome IX Conference
Poster: Large Insert Libraries, Gene Isolation, Etc.
P02_31.html
One approach in reverse genetics to identify the function of a gene, is the selection of insertion mutants. Often, insertions in single genes belonging to large gene families do not cause any visible phenotype due to redundancy. Therefore, such single insertion mutants may have to be combined in order to obtain a full loss-of-function phenotype, shifting single gene research towards the gene family level. Classically, an insertion screening is carried out by combining a gene-specific primer with a T-DNA/transposon-specific primer in a PCR: Positives are detected by hybridizing blotted PCR products with a gene-specific probe. When screening for insertions into genes belonging to large and highly homologous gene families, cross-hybridization of different family members can seriously complicate downstream analysis. This problem might be circumvented by choosing primers and probes in non-conserved regions, but then a separate screening for each individual family member is required, making the whole screening labor intensive. Therefore, we developed a screening method that omits the hybridization step in classical methods. Identification of positives is done by direct sequencing of bands amplified from pools of insertion lines. Since the identification is sequence based, even highly homologous genes can be easily distinguished. This approach allows to simultaneously identify insertion events in different family members, and has the additional advantage to identify yet unknown (sub)family members through their corresponding insertion mutant. We used this method in order to identify insertions mutants into known and yet unknown Petunia MADS-box genes.