Genotyping β-globin Mutations (Hb S, Hb C, Hb E) by Multiplexing Probe Color and Melting Temperature

Abstract

Human β-globin has over 50 known mutations in exon 1 causing various hemoglobinopathies (1,2). The mutations include base pair substitutions, deletions and splicing defects. Hemoglobin S and C, (codon 6) and E (codon 26) are base pair substitutions that occur often enough to allow for routine screening (Hb S occurs at an allele frequency of 1:400 in African American’s)(3). High performance liquid chromatography (4) and isoelectric focusing (5) are routinely used for primary patient screening. However, phenotypic screening does not always necessarily coincide with the genotype, for example Hb S, Hb G_Norfolk and Hb_{Fort-de-France} all have the same isoelectrofocusing point (6). Genotyping can be done by allele specific amplification (7), DNA amplification followed by restriction digestion (8) or by fluorescently labeled allele specific primers and gel based detection with color photography (9). These methods require hours to days for completion. Recently, genotyping by determining the melting temperature (T_m) of hybridized fluorescently-labeled oligonucleotide probes was reported (10,11). We have extended the power of this technique by multiplexing different colored probes to simultaneously genotype multiple alleles (12). This procedure has been applied to homogenous genotyping of hemoglobin S, C, and E in a single tube by melting curve analysis by using different colored probes and T_m multiplexing.