Light cycler 480 gene scanning software

Leaf total DNA of the corresponding accessions were directly extracted using the cetyltrimethylammonium bromide (CTAB) method described by Lutz et al., [58 (link)] and then subjected to genotyping analysis. High Resolution Melting (HRM) experiments were performed in 98/384-well plates using the Roche LightCycler 480® High Resolution Melting PCR Master Mix and analyzed by the LightCycler 480® Gene Scanning Software. Kompetitive Allele Specific PCR (KASP) analysis used for variant validation and haplotype dissection were performed using KASP Master mix according to the company’s protocols (LGC Genomics, Teddington, Middlesex, UK) on the Roche LightCycler 480® System.

Genomic DNA was extracted from peripheral blood samples using BioTeke DNA Blood kit according to the manufacturer's instructions and was stored at −80°C.
PCR components included Master Mix (520 ul; Roche) containing FastStart Taq DNA polymerase reaction buffer, dNTPs and HRM dye; 10.4 ul each forward and reverse primers of rs246871/rs31223/rs25855 respectively; 25 nM MgCl₂ (Roche). Wild-type and mutant homozygotes were distinguished by spiking samples with a known genotype obtained by sequencing previously before PCR. Unknown genomic DNA (10 ng) was used as template. H₂O (Roche) was added to bring the final reaction volumes to 1040 ul.
PCRs were conducted in 96-well plates in 10 ul-volumes using the following touchdown PCR cycling and HRM conditions: initiation with a 10-min hold at 95°C, 46 cycles of 95°C for 15 s, touchdown cycling (decreasing 1°C/cycle), annealing in the range of 65–55°C for 10 s, and 72°C for 20 s. Following amplification, samples were heated to 95°C for 1 min and then cooled to 40°C for 1 min encouraging heteroduplex formation. HRM curve data were obtained by melting over the range 65–95°C at a rate of 15 data acquisitions per 1°C. Results were analyzed using Light Cycler 480 Gene Scanning software (Roche Applied Science, Germany).

DNA was screened for the rs7975232, rs1544410, rs731236, rs10735810, and rs11568820 polymorphisms in the VDR gene using TaqMan® assays on a Roche LC480 (Roche Diagnostics, Penzberg, Germany). Primers were designed using Primer Premier 5 (Premier Biosoft Inter, Palo Alto, USA) (Table 1) and synthesized by Shanghai Shenggong Co. (Shanghai, China). PCR reaction mixtures and conditions were designed according to the manufacturer's instructions (B639274 BBI, Shanghai Shenggong Co, Shanghai, China). Genotyping fidelity was verified using positive control subjects in each 384-well plate and re-running ≥5% of samples, which generated 100% concordant results. Genotype identification was performed using LightCycler480 Gene Scanning Software (Roche Diagnostics, Penzberg, Germany) and was blinded, with no information on the phenotypes of the subjects available to the individual conducting genotyping. Identified genotypes of rs10735810 were CC, CT, and TT; those of rs1544410 were AA, AG, and GG; those of rs731236 were TT, TC, and CC; those of rs7975232 were AA, AC, and CC; and those of rs11568820 were GG, AG, and AA.

Collected data were analyzed by the Light Cycler 480 Gene Scanning software v1.2 (Roche Diagnostics). Software programs used the following analysis method: normalization by selecting linear regions before and after the melting transition, temperature shifting by selecting the threshold, then automatic grouping by calculation. The exact same setting of the normalization was used for all experiments. The genotype of each subset was defined, according to known genotypes of controls.