Lactase persistence genotyping: rapid detection of seven sequence variants in a single tube with melting curve analyses.

BACKGROUND: Lactase persistence is an inherited autosomal dominant trait that confers the ability to digest lactose after weaning. Lactose persistence is caused by single nucleotide variants in a regulatory element for the lactase gene (LCT). In Europeans, lactase persistence is associated with the nucleotide variant LCT -13910C>T. Ethnic groups from Africa and the Arabian Peninsula have other lactase persistence variants in close proximity to the -13910C>T variant. Current hybridisation probe analysis methods have been limited by the inability to detect all the known nucleotide variants. The aim of this study was to devise a method based on hybridisation probes and melting curve analysis for rapidly detecting LCT variants.

METHODS: We designed hybridisation probes for producing melting curve profiles that could identify seven LCT nucleotide variants: -13907C>G, -13910C>T, -13913T>C, -13914G>A, -13915T>G, -14009T>G, and -14010G>C. To validate the assay, we generated plasmid standards of all the alleles and mixed them to create artificial heterozygote samples.

RESULTS: All genotypes could be detected in a single tube assay. Rare genotypes were confirmed in a second assay with probes that had different complementary sequences.

CONCLUSIONS: This assay can be used for rapidly genotyping lactase persistence in multi-ethnic populations.