Detection of human DNA mutations with nonradioactive, allele-specific oligonucleotide probes.
We describe a method of detecting human DNA mutations with nonradioactive, biotinylated allele-specific oligonucleotide probes. This method can detect seven different mutations in the butyrylcholinesterase, cystic fibrosis, and N-acetyltransferase genes under identical assay conditions. This indicates that it may be used to detect mutations responsible for a wide variety of genetic diseases and pharmacogenetic conditions. The method involves first amplifying selected DNA fragments by the polymerase chain reaction and dot blotting the amplified DNA in duplicate onto small nitrocellulose squares. Each dot blot is then hybridized in individual wells containing a tetramethylammonium chloride solution with short biotinylated probes specific for either the normal or mutant allele. Successfully hybridized probes are detected by a simple colorimetric reaction using an avidin-alkaline phosphatase conjugate, which yields a very strong, clear signal. DNA from homozygous normal or mutant individuals hybridizes only to the normal- or mutant-specific probes respectively, while DNA from heterozygous individuals hybridizes equally well with both probes. These results can be easily interpreted to assign a genotype to the sample DNA. This method is amenable to automation, and may be useful in clinical laboratories for diagnosis of a wide variety of DNA mutations responsible for unusual reactions to drugs and environmental chemicals.
Hajra
A
A
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0572.
Sorenson
R C
RC
La Du
B N
BN
D000483:Alleles; D001191:Arylamine N-Acetyltransferase / Q000235:genetics; D001483:Base Sequence; D001710:Biotin; D002091:Butyrylcholinesterase / Q000235:genetics; D003550:Cystic Fibrosis / Q000235:genetics; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes
D015345:Oligonucleotide Probes; D001710:Biotin; D004247:DNA; D001191:Arylamine N-Acetyltransferase; D002091:Butyrylcholinesterase