Rapid β-Globin Genotyping by Multiplexing Probe Melting Temperature and Color

[摘要] Single-nucleotide polymorphisms have been identified by many different experimental approaches. The human β-globin gene has been genotyped by several methods, including PCR followed by restriction digestion (1)(2), denaturing gradient gel electrophoresis (3), allele-specific amplification during PCR, and the ligase chain reaction (4). These methods require several hours and sometimes days for diagnosis. Recently, rapid-cycle PCR has been combined with real-time fluorescence monitoring to detect mutations by fluorescent probe melting point analysis for homogeneous genotyping in <1 h. Fluorescent melting point analysis is a technique that detects mutations by differences in the melting temperature ( T m) of fluorescent oligonucleotides hybridized to different alleles (5)(6)(7). Probes of a single color are usually used for genotyping. Four alleles at two different loci have been genotyped by multiplexing probe T ms of a single color (8). However, there is a limit to how many alleles can be distinguished by differences in T m. The ability to use multiple colored probes along with T m would greatly extend the power of monitoring PCR with fluorescence by allowing greater numbers of loci to be screened for mutations in one reaction. Exon 1 of the β-globin gene has >50 mutations, which produce various hemoglobinopathies (9). Hemoglobins S, C, and E are common and are routinely screened. Hemoglobin C (Hb C) results from a G-to-A mutation in the first nucleotide of codon 6, whereas hemoglobin S (Hb S) arises from an A-to-T mutation in the second nucleotide of this codon. Hemoglobin E (Hb E) results from a G-to-A mutation in the first nucleotide of codon 26. The close proximity of these three mutations allowed us to design a probe system that discriminated all genotypes using T m and two probe colors.The human β-globin gene sequence (GenBank accession no. U01317 …

[学科分类] 过敏症与临床免疫学