Melting Temperature Assay for a UGT1A Gene Variant in Gilbert Syndrome

Abstract

For detection of nucleotide polymorphisms or microinsertions/deletions, an optimal genotyping method would be rapid and economical. Newer, homogeneous, and automated genotyping methods with fluorescent oligonucleotide probes (1)(2)(3) can be expensive, and their use is difficult to optimize. Here we demonstrate the application of melting temperature ( T m) analysis (4) in a study of the UGT1A gene in a family with Gilbert syndrome. Both amplification and T m analysis are performed on the same fluorescence-detection thermocycler, the GeneAmp 5700 Sequence Detection System (PE Biosystems). The single-tube assay uses a specific primer set, with a high annealing temperature, and a fluorescent dye that can detect double-stranded DNA product. Because the temperature at which double-stranded templates dissociate depends largely on product length, GC content, and sequence structure, the PCR product has a distinct T m, depending on base substitutions, single point mutations, or insertions/deletions. Soon after the last amplification cycle, the reaction is slowly cooled by 1 °C steps from 95 °C to 60 °C. Detection follows this cooling process, with the related SYBR green emissions reflecting the amplicon renaturation kinetics. Analysis of the resulting melting profiles allows determination of insertions and, hence, of the corresponding genotype of the genomic template. The most consistent feature of Gilbert syndrome is a deficiency in bilirubin glucuronidation. The UGT1A gene is responsible for bilirubin catalytic enzyme production. The lower expression of the UGT1A gene is related to a TA insertion in the TATA box of the gene promoter (5). The resulting PCR product differs from the wild-type gene in having a two-base insertion. This T m analysis of PCR products allowed discrimination between …