Dnaengine thermo cycler

To assess KRAS exon 2 mutational status, 50 ng of template DNA was used for standard PCR amplification using 0.12 μM of each primer (forward: 5′-GGTGGAGTATTTGATAGTGTA-3′; reverse: 5′-TGGACCCTGACATACTCCCAAG-3′), 2 mM of MgCl₂ (NzyTech, Lisbon, Portugal), 0.8 mM of dNTP Mix (NzyTech, Lisbon, Portugal), and 0.15 units of NZYTaq II DNA Polymerase (NzyTech, Lisbon, Portugal) in a final reaction volume of 20 μL. Reactions were performed on a DNA Engine® thermocycler (Bio-Rad, Hercules, CA, USA) as follows: 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 61 °C for 30 s for tumor samples and 53 °C for 30 s for plasma samples, and 72 °C for 20 s. For plasma samples, four independent amplification reactions were performed for each sample, which were later concentrated using the SpeedVac Concentrator (Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in 15 μL of DEPC-treated water.
PCR products were then direct sequenced at STAB VIDA (Setubal, Portugal), and the chromatograms analyzed using FinchTV software (Geospiza, Inc) for sequence characterization and identification of possible mutations in codon 12.

DNA-binding reactions were set up in 20 μl with varying concentrations of PfMCM_N (0–54 μM) and 160 nM 5′-fluorescein-labeled T40 ssDNA (Sigma-Aldrich, St. Louis, MO) in 20 mM HEPES, pH 7.6, 200 mM NaCl, 5 mM MgCl₂, and 5 mM βME. Reactions were incubated at 25°C in a BioRad DNA Engine thermocycler for 30 min. Loading buffer (2.5 mg/ml bromophenol blue and 40% sucrose; 5 μl) was added, and 5 μl were loaded in a 4–20% 1X TBE gradient PAGE gel (BioRad, Berkeley, CA) and run at 100 V for 105 min. Gels were imaged by a Fuji LAS-4000 with an 8 s exposure and a SYBR-Green filter. The fluorescence intensities of bands for the free and bound species were quantified with MultiGauge (GE Healthcare, Piscataway, NJ) and fit to two simultaneous equations with Prism (GraphPad Software, La Jolla, CA): $\text{I}\left(\text{free}\right)/{\text{I}}_{\text{0}}={\text{K}}_{\text{half}}^{\text{h}}\text{/}({\text{K}}_{\text{half}}^{\text{h}}+{{[\text{MCM}}_{\text{N}}]}^{\text{h}}) ; \text{I}\left(\text{bound}\right)\text{/}{\text{I}}_{\text{0}}={{[\text{MCM}}_{\text{N}}]}^{\text{h}}/({\text{K}}_{\text{half}}^{\text{h}}+{{[\text{MCM}}_{\text{N}}]}^{\text{h}})$ to determine the concentration of half-binding (K_half) and a hill coefficient (h). The dsDNA EMSAs were identical except that they included a 26-mer dsDNA substrate and a different concentration range of PfMCM_N (0–20 μM). The dsDNA substrate was prepared by annealing two oligos (5′-[Fluorescein]-ATGGCAGATCTCAATTGGATATCGGC-3′ and 5′-GCCGATATCCAATTGAGATCTGCCAT-3′, Sigma-Aldrich) followed by purification on a gel filtration column (GE Healthcare Superose 12 10/300).