One step real time pcr system

Thermal shift assays were performed as described (Hill et al., 2019 (link)) using the Step One Real-Time PCR System^TM (Thermo Fisher Scientific) and Step One^TM software. The final volume for the assay was 20 μl and contained 50 mM HEPES (pH 8.0), SYPRO Orange Dye (Sigma-Aldrich) (4X) and 0.02 mg/ml purified protein, in the absence and presence of 1 mM FBP, 0.25 mM AMP, and 10 μM PLP. A control with no protein was also performed for all the samples. A continuous temperature increase from 20.0 to 99.0°C was scanned every 0.4°C in a ramp increment of 1.5°C per minute. Scans were run in triplicates and averaged. The unfolding temperatures (T_m) of the proteins were measured using the minimum of the third derivative of the scanned fluorescence vs. temperature (d³F/dT³). Smoothing and differentiation of the thermograms were performed using a twenty-five-point Savitzky–Golay algorithm to determine the T_m (Savitzky and Golay, 1964 (link)). Traditionally, T_m has been determined by the minimum of the negative of the first derivative (–dF/dT). However, we preferred the use of higher derivatives because it is well-documented for spectrometry that it removes drifts, interferences, and enhances sharper peaks (Butler, 1979 ). Calculated T_m did not vary, but the graphical comparison of different thermal melting curves was clearer using d³F/dT³.

The unfolding temperature of proteins (T_m) was measured in the presence and absence of ligands to evaluate their ability to bind to the protein, stabilize it, and consequently shift the T_m.⁴⁰, ⁴¹ Solutions of (4×) SYPRO Orange fluorescence dye (Sigma‐Aldrich), 20 mM HEPES buffer (pH 7.5), 0.02 mg/ml protein, and ligands as indicated were added in a total volume of 20 μl. The solution was placed into the wells of a 96‐well real‐time PCR plate and covered with sealing tape. The equipment used was the Step One Real‐Time PCR System^TM from Thermo Fisher Scientific (Waltham, MA) with its software Step One^TM. The temperature was scanned from 25°C to 99°C and the fluorescence changes in the plate wells were recorded.⁴¹ The unfolding temperatures (T_m) of the proteins were measured using the minimum of the negative of the first derivative of the scan fluorescence versus temperature (−dF/dT).

Genomic DNA of BC, OC and PC patients were collected using buccal swabs and extracted through MagPurix instrument and Forensic DNA Extraction Kit (Zinexts Life Science Corp.- CodZP01001) according to the manufacturer’s protocol. NGS was executed by the Ion Torrent S5 system (Thermo Fisher Scientific, Waltham, MA, United States) after automatic library preparation using Ion Chef (Thermo Fisher Scientific, Waltham, MA, United States). Ion Chef consists of fragmentation and adapter ligation onto the PCR products, clonal amplification. The DNA libraries were quantified with Real-Time Step One PCR System (Thermo Fisher Scientific, Waltham, MA, United States) and the prepared samples were loaded onto an Ion 530™ chip by Ion Chef (Thermo Fisher Scientific, Waltham, MA, United States). Ion S5™ Plus (Thermo Fisher Scientific, Waltham, MA, United States) instrument was used for the sequencing. Specific plugins as “SampleId” and “Coverageanalysis” were used for NGS data analysis on the Torrent Suite 5.14.0 platform. The uniformity of base coverage was over 98% in all batches, and base coverage was over ×20 at all target regions. This NGS method cannot detect variations outside the +/−10 nucleotide coding sequence.