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. 2019 Sep;21(5):782–789. doi: 10.1016/j.jmoldx.2019.04.006

Figure 2.

Figure 2

AS-PCR results. A:HLA-A*32:01–positive (+) and non–HLA-A*32 (−) samples were amplified by PCR using the following validated primers: HLA 32-88F forward primer and HLA032R2 reverse primer to amplify the HLA-A*32 allele. GALC-F and GALC-R primers were used to amplify the internal control housekeeping gene in a multiplexed reaction. PCR products were run on a 1% agarose gel containing 0.2 μg/mL ethidium bromide at 115 V for 30 minutes. The gel was visualized by a transilluminator (ChemiDoc XRS+; Bio-Rad Laboratories). HLA-A*32:01–positive samples show two bands of 157 bp (HLA-A*32:01 product) and 352 bp (GALC product). Non–HLA-A*32 samples show only one band of 352 bp (GALC product). B: Tm peaks of both HLA-A*32:01 and GALC housekeeping gene are shown by an arrow. Tm peaks for the HLA-A*32:01 allele were clearly separate from the GALC Tm peak following melt curve analysis. C: Melting peaks for a subset of the 458 samples tested in a real-time PCR with PowerUp SYBR Green are shown. Tm peaks of HLA-A*32:01 and non–HLA-A*32:01 alleles are shown by arrows. HLA-A*32:01–positive samples show double Tm peaks at 88.5°C ± 0.0°C (range: 88.50°C to 88.50°C) for the HLA-A*32:01 allele and 76.05°C ± 0.03°C (range: 76.00°C to 76.50°C) for GALC. Non–HLA-A*32 allele samples show a single Tm peak at 76.07°C ± 0.01°C (range 75.50°C to 76.50°C) for GALC. D: Melting curves for a subset of the 458 samples tested in real-time PCR. Data are expressed as means ± SEM. n = 30 (C, HLA-A*32:01–positive samples). RFU, relative fluorescence units; Tm, melting point.