Scanning detection of mutations in human ornithine transcarbamoylase by chemical mismatch cleavage.
The mutations in X chromosome-linked diseases are frequently heterogeneous, and yet only a small fraction can be detected by Southern analysis. We therefore adapted the chemical cleavage method of Cotton et al. [Cotton, R. G. H., Rodrigues, N. R. & Campbell, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 4397-4401] and the polymerase chain reaction to rapidly scan for point mutations in X chromosome-linked ornithine transcarbamoylase (carbamoyl-phosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) deficiency. This simple heteroduplex mapping method identified different mismatch sites in polymerase chain reaction-amplified liver cDNA from five unrelated ornithine transcarbamoylase-deficient patients. The predicted sequence alteration was confirmed by DNA sequencing in all five patients and indicated a likely disease-causing mutation in four of these patients. In one atypical ornithine transcarbamoylase-deficient patient a sequence alteration compatible with a cDNA polymorphism was found. One family was studied in detail. Female-carrier detection was performed by chemical cleavage of amplified genomic DNA and verified by allele-specific oligonucleotide hybridization. This mutation scanning approach is simple, sensitive, and applicable to many genetic loci.
Grompe
M
M
Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030.
Muzny
D M
DM
Caskey
C T
CT
D000328:Adult; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005260:Female; D005784:Gene Amplification; D005796:Genes; D006801:Humans; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009954:Ornithine Carbamoyltransferase / Q000235:genetics*; D010375:Pedigree; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D014960:X Chromosome
D003062:Codon; D012313:RNA; D004247:DNA; D009954:Ornithine Carbamoyltransferase; D004259:DNA-Directed DNA Polymerase