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

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Abstract
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 Tm) 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 Tms of a single color (8). However, there is a limit to how many alleles can be distinguished by differences in Tm. The ability to use multiple colored probes along with Tm 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 Tm and two probe colors.