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 designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. 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 same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.
Hacia
J G
JG
National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Sun
B
B
Hunt
N
N
Edgemon
K
K
Mosbrook
D
D
Robbins
C
C
Fodor
S P
SP
Tagle
D A
DA
Collins
F S
FS
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins; D005091:Exons / Q000235:genetics; D005260:Female; D016045:Human Genome Project; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics*; D008297:Male; D009154:Mutation / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D025521:Tumor Suppressor Proteins
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human