Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were de signed to screen For all possible heterozygous germ-line mutations in the 9 .17-kb coding region of the ARI gene. A strategy For rapidly developing mul tiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exon s. Improved algorithms For interpreting data From two-color experiments, wh ere reference and test samples are cohybridized to the arrays, were develop ed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these sam e samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuabl e role in high throughput sequence analysis of complex genes.