Detection of single nucleotide substitution by competitive allele-specific short oligonucleotide hybridization (CASSOH) with immunochromatographic strip.
Recent advances in human genome research have revealed that genetic polymorphisms, such as single nucleotide polymorphisms (SNPs), are closely associated with susceptibility to various common diseases and adverse drug reactions. Also, numerous mutations responsible for a number of genetic diseases have been identified. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We have devised a novel low-tech method for the detection of a single nucleotide substitution using competitive allele-specific short oligonucleotide hybridization with immunochromatographic strip. The gene of interest is PCR-amplified, hybridized to an allele-specific short oligonucleotide probe in the presence of a competitive oligonucleotide, and subjected to chromatography using a DNA test strip at room temperature. The genotype is unambiguously determined by the presence or the absence of visible purple lines on a strip. Feasibility of the method was demonstrated by the detection of a prevalent disease-causing mutations in glycogen storage disease type Ia (G6PC), medium-chain acyl-CoA dehydrogenase deficiency (ACADM), non-ketotic hyperglycinemia (GLDC), and clinically important polymorphisms in the CYP2C19 gene and the aldehyde dehydrogenase 2 gene (ALDH2). The procedure does not demand either technical expertise or expensive instruments and is readily performed in local clinical laboratories. The result is obtained within 10 min after PCR. This rapid and simple method of SNP detection may be used for point-of-care genetic diagnosis with potentially diverse clinical applications. Hum Mutat 22:166-172, 2003.
Matsubara
Yoichi
Y
Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan. ymats@mail.cc.tohoku.ac.jp
Kure
Shigeo
S
D000483:Alleles; D001667:Binding, Competitive / Q000235:genetics*; D002845:Chromatography / Q000379:methods*; D004247:DNA / Q000235:genetics; D005189:False Positive Reactions; D005240:Feasibility Studies; D005838:Genotype; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009711:Nucleotides / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*
D009711:Nucleotides; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D004247:DNA