Amplitaq gold dna polymerase

Target sequences were quantitated by TaqMan methodology based quantitative real-time PCR (RT-PCR). The custom made, gene specific pre-validated TaqMan Gene Expression Assays (Assays-On Demand IDs: TP53: Hs00153408_m1; KRT1: Hs00196158_m1; KRT10: Hs00166289_m1; KRT19: Hs00761767_m1; MMP9: Hs00957562_m1) and the TaqMan Gene Expression Master Mix containing AmpliTaq Gold® DNA Polymerase were purchased from Thermo Fisher Scientific, Woolston, UK. Gene expression measurements were carried out with an ABI Prism 7900HT Sequence Detection System (Thermo Fisher Scientific, Woolston, UK) according to the manufacturer’s instructions; briefly 4 ng of cDNA was diluted into a total of 20 μL reaction volume containing 10 μL TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) with AmpliTaq Gold DNA Polymerase and the target gene specific TaqMan Gene Expression Assay mix. Amplification was performed for 40 cycles, including denaturation at 95°C for 15 seconds, annealing at 60°C and extension at 72°C for 60 and 30 seconds, respectively. Relative quantification of mRNA expression levels of the target genes was performed using the comparative threshold method using the PPIA gene as an endogenous reference [Assays-On Demand ID: Hs99999904_m1 (Thermo Fisher Scientific)].

PCRs were performed using AmpliTaq Gold DNA polymerase (Applied Biosystems by Life Technologies). Products were run on an agarose gel and purified using the A’SAP PCR cleanup kit (Arctic Zymes) and Sanger sequenced at FIMM. Sequences were analyzed manually and with Mutation Surveyor (v4.08) (Softgenetics). PCRs from archival samples were performed in at least three replicates. For cDNA synthesis, RNA was reverse transcribed using random primers and Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega). The primers are listed in Supplementary Material, Table S9 and were designed with Primer3web (v4.0.0) (34 (link),35 (link)).

PCR amplification was carried out in 20-μl volumes containing 10–20 ng of the extracted genomic DNA, 1 × PCR buffer (Applied Biosystems), 2.0 mM MgCl₂, 0.2 mM of each dNTP, 3 pmol of each primer, and 0.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems). We performed PCR amplification using 23 overlapping fragments of the complete mtDNA^{40 (link)} and variable portions of the control region primers, Cerv.tPro, and CervCRH^{41 (link)}. The thermal cycling conditions for both the primer pair was as follows: an initial hot start at 95 °C of 10 minutes, followed by 35 cycles at 95 °C for 45 seconds, 55 °C for 45 seconds and 72 °C for 1 minute, with a final extension of 72 °C for 15 minutes. The efficiency and reliability of the PCR reactions were monitored using control reactions. The PCR products were electrophoresed on 2% agarose gel and visualized under UV light in the presence of ethidium bromide dye. The amplified PCR products were treated with Exonuclease-I and Shrimp Alkaline Phosphatase (USB, Cleveland, OH) for 15 minutes each at 37 °C and 80 °C, respectively, to remove any residual primer. The purified fragments were sequenced directly in an Applied Biosystems Genetic Analyzer 3500XL from both primers set using a BigDye v3.1 Kit.

Nine different combinations, between 0.2 μM and 0.9 μM, of the forward and reverse primers and five different concentrations of probe ranging from 0.05 μM to 0.25 μM were tested with DNA (600 pg) from S. noxia reference strain ATCC 51893 to determine the optimal amplification signal. The PCR cycling parameters were as follows: initial incubation step of 50 °C for 2 min, denaturation of the template DNA at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. PCR conditions were: 1X TaqMan universal PCR master mix containing AmpErase® UNG (uracil-N-glycosylase), AmpliTaq Gold DNA polymerase, deoxynucleoside triphosphates, passive internal reference [ROX™ dye], and optimized buffer components (Applied Biosystems), primers (SNF1 and SNR1or SNF2 and SNR2) to a final concentration of 0.9 μM, probe to a final concentration of 0.2 μM, and 5 μl of template DNA. Sterile nuclease-free water (Promega, Madison, WI) was used to adjust the volume of each reaction to 25 μl. The analyses were performed in 96-well plates using a 7900HT Fast real-time PCR system instrument (Applied Biosystems) on standard mode. Negative controls containing 5 μl of nuclease-free water instead of DNA were included in each run to detect any DNA cross-contamination. DNA extracted from S. noxia served as positive control in each PCR run.

To confirm the results of RT-PCR, mRNA expression was also analyzed using an iCycler iQ Multi-Color Real Time PCR Detection System (Bio-Rad, Hercules, CA) with SYBR Green (Molecular Probe, Eugene, OR). The primer sequences used for real-time PCR and expected product size were described above. In brief, 1 μL of cDNA was added in a 24 μL reaction mixture containing 0.5× SYBR Green, 1× PCR buffer, 0.6 mM MgCl₂, 0.4 mM dNTP, 0.5 mM primer sets and 0.625 U Amplitaq gold DNA polymerase (Applied Biosystems, Foster City, CA). The cycling conditions for all genes were as follows: preincubation at 50°C for 2min, 95°C for 4min, followed by 55 cycles of denaturation at 95°C for 20 s, annealing at 65°C for 30 s and extension at 72°C for 30 s. At the completion of cycling, melting curve analysis was performed to establish the specificity of the PCR product. The expression level of cDNA of each candidate gene was internally normalized using GAPDH. The relative quantitative value was expressed by the 2^-ΔΔCT method, representing the amount of candidate gene expression with the same calibrators. Each experiment was performed in duplicate and repeated three times.

Seven pairs of published oligonucleotide primers were used to detect the virulence genes using PCR. The individual PCRs, for each virulent gene, were set up in a 25 µL, which consisted of 12.5 µL AmpliTaq Gold® 360 PCR Master Mix (AmpliTaq Gold® DNA Polymerase 0.05 units/µL, Gold buffer 930 mM Tris/HCl pH 8.05, 100 mM KClO, 400 mM of each dNTP and 5 mM MgCl₂) (Applied Biosystems, California, US). Then, 2.5 mM of each primer, 2 µL of template DNA and ddH₂O were added to make the final volume. Test DNA was replaced with 5 µL of sterile nuclease-free water as negative control. Cycling conditions for PCR as well as the information of the published primers used are detailed in Table 1.