7500 fast real time pcr thermal cycler

The molecular diagnostic assay we have developed is based on nucleic acid amplification by PCR and TaqMan chemistry. It targets the pathogenic variant c.262del within the CYP27B1 gene, which causes VDDR1A. The variant is detected by two sets of specific primers and probes we have designed and validated. The two sets of primers and TaqMan probes were directed against the pathogenic variant c.262del and independently tested to improve the analytical validity of the assay. The sequences of the probes and primers used are shown in Supplementary Table 1. For each amplification reaction, 1.75µL of sterile water, 3.10µL of GTXpress Master Mix, 0.15µL of TaqMan assay, and 1.2µL of samples were used. Analysis was carried out in a 96-well plate and samples were amplified on a 7500Fast Real-Time PCR thermal cycler from Applied Biosystems Inc. The amplification conditions were as follows: 1. 60°C for 1 minute with fluorescence acquisition, 2. 95°C for 20 seconds, 3. 95°C for 3 seconds, and 60°C for 30 seconds with fluorescence acquisition (repeat step 3 forty times), 4. 60°C for 1 minute with fluorescence acquisition.

Real-time PCR with HRM analysis was performed in a total volume of 10 μL using a 7500 Fast Real-time PCR thermal cycler (Applied Biosystems). The reactions contained 5 μL of MeltDoctor^TM HRM Master Mix (Applied Biosystems), 0.6 μL of each forward and reverse primer, PCR additives, 1 μL of DNA and topped up to 10 μL with double-distilled water. The thermal cycling conditions involved enzyme activation at 95 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 15 seconds and 1 minute of annealing/extending at 60 °C. For the melt curve analysis, the thermal cycling conditions started with denaturation at 95 °C for 10 seconds, annealing at 60 °C for 1 minute, HRM at 95 °C for 15 seconds and finally annealing at 60 °C for 15 seconds. DNA amplification was checked using Applied Biosystems 7500 Software version 2.0.6. The melt curve analysis was performed using the Applied Biosystems HRM Software version 2.0.1.

For Experiment 2, absolute telomere length (aTL) was measured using methods nearly identical to those used in Experiment 1. Because telomere length quantification was performed by independent experimenters for Experiment 1 and Experiment 2, there were some minor differences in methodology: First, real-time PCR was run in triplicate on the Applied Biosystems 7500 Fast Real-Time PCR thermal cycler (Waltham, MA, USA) for Experiment 2. Second, Experiment 2 DNA samples used for real-time PCR were slightly more concentrated (1.5 ng/µL). Finally, raw data (not normalized to a plate control) were used for Experiment 2 statistical analyses. Note that another form of plate normalization, the inclusion of plate as a random statistical factor, was still performed for Experiment 2.