Duplex Probe-Based Fluorescence Melting Curve Analysis for Simultaneous Genotyping of rs1126728 and rs11208257 in the Phosphoglucomutase-1 Gene

Background: Phosphoglucomutase-1 (PGM1) is an enzyme that plays important roles in glycolysis, glycogen metabolism, and glycosylation. The PGM1 gene harbors two common nonsynonymous single-nucleotide variants (rs1126728 and rs11208257), which result in four functional PGM1 phenotypes. Correlations between PGM1 polymorphisms and sev-eral pathological conditions have been suggested. Methods: To identify the rs1126728 and rs11208257 concurrently, a fluorescence melting curve analysis (FMCA) was developed that utilizes two distinct dual-labeled fluorescence probes. Two distinct Taq polymerases, one with and one without 5’-3’exonuclease activity, were compared. This method was then applied to 95 unrelated Japanese subjects. Results: Both Taq polymerases, with and without 5’-3’exonuclease activity, were found to be sufficiently functional. Furthermore, the results of the FMCA using both Taq polymerases were compared with the direct Sang-er sequencing results of PCR products from the 95 Japanese subjects, demonstrating 100% concordance. Conclusions: The duplex probe-based FMCA developed in this study is useful for examining the association between rs1126728 or rs11208257 and a range of pathological conditions using a relatively large number of subjects.