Multiplex genotyping by melting analysis of loci-spanning probes: beta-globin as an example

Multiplexing genotyping technologies usually require as many probes as genetic variants. Oligonucleotides that span multiple loci--loci spanning probes (LSProbes)--hybridize to two or more noncontiguous DNA sequences present in a template and can be used to analyze multiple variants simultaneously. The intervening template sequence, omitted in the LSProbe, creates a bulge-loop during binding. Melting temperatures of the probe, monitored by fluorescence reading are specific to the presence or absence of the mutations. We previously described LSProbes as a molecular haplotyping tool and apply here the principle to genotype simultaneously three mutations of the beta-globin gene responsible for the corresponding hemoglobinopathies. Analysis with both labeled and unlabeled LSProbes demonstrate that the four possible alleles studied (WT, HbS, HbC, and HbE) are identifiable by the specific melting temperatures of the LSProbes. This demonstrates that, in addition to their haplotyping capabilities, LSProbes are able to genotype in a single step, loci 58 nucleotides apart.