Detection of four beta-thalassemia point mutations in Iranians using a PCR-ELISA genotyping system.
Development of molecular techniques with analytical capability of mutation detection can realize the medical diagnosis of diseases and improve people's health. beta-Thalassemia is one of the most prevalent genetic disorders in Iran and using a simple and rapid test in laboratories for the mass screening and prenatal diagnosis is essential. Here, we described a simple method for rapid detection of four common beta-thalassemia point mutations in Iranians (IVS-II-1 (G-->A), IVS-I-5 (G-->C), FSC 8/9 (+G), IVS-I-110 (G-->A)) using a PCR-ELISA genotyping system. After DNA isolation from whole blood, a segment of beta-globin gene was amplified by DIG-labeling PCR. The DIG-labeled PCR amplicons were denatured and added to biotinylated normal probe (for normal gene allele) and mutant probe (for mutant gene allele). The hybrids were detected by colorimetric ELISA method. The optical densities obtained using normal and mutant probes with heterozygous PCR products were very similar. The optical densities obtained using mutant probes were higher than normal probes with homozygous PCR products. In vice versa, the optical densities obtained using normal probes were higher than mutant probes with normal PCR products. All the results demonstrated that the PCR-ELISA has similar specificity in comparison to the amplification refractory mutation system.
Gill
Pooria
P
Research Center for Molecular Biology, Baqyiatallah Medical Sciences University, Tehran, Iran. pooriagill@yahoo.com
Forouzandeh
Mehdi
M
Eshraghi
Naser
N
Ghalami
Mostafa
M
Safa
Majid
M
Noori-Daloii
Mohammad-Reza
MR
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005838:Genotype; D006801:Humans; D007492:Iran; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017086:beta-Thalassemia / Q000235:genetics*