A microarray approach for identifying mutated genes

This study was performed to evaluate if microarray technology can identify genes using their transcriptionally defective mutant alleles. Three barley (Hordeum vulgare L.) mutant strains, xantha-h57, xantha-f27 and xantha-g28, with mutations in the genes encoding the three subunits of the chlorophyll biosynthetic enzyme magnesium chelatase, were used in a reconstruction experiment. The mutation xantha-h57 prevents transcription of Xantha-h mRNA. Microarrays were prepared by robotic spotting of PCR products from 968 clones at 1760 positions. Most of the materials were from cloned expressed sequence tags (ESTs). The barley Xantha-h gene was printed at six positions in duplicate. Crude bacterial lysates as well as purified plasmids were used as template in the PCR reactions. The bacterial lysates did not affect the printing quality of the DNA. This simplification is important, as it increases the throughput capacity and decreases costs. cDNA from the three mutants and the wild-type strain were differentially labeled with fluorescing nucleotides. Labeled cDNA from one mutant was mixed in equal amounts with labeled cDNA of another mutant or wild type and competitively hybridized to the microarrayed clones. The combination of labeled cDNA from xantha-h57 with that of xantha-f27 or xantha-g28 specifically highlighted the positions representing the Xantha-h gene on the microarrays. Therefore, we regard this experiment as a demonstration that microarrays provide a very promising method for screening large DNA libraries in order to clone genes known only through their mutant phenotype. This opens up a new way of using the microarray technology for cloning genes from eukaryotes with complex genomes for which genome sequencing is unlikely to proceed. Our results also put the many available plant mutant collections in focus as treasuries for gene hunters.