Hot start taq dna polymerase

Single PCR was conducted under the following conditions: the final volume was 25 µL, containing 2.5 µL 10× buffer (Bioneer, Daejeon, Korea), 0.2 mM dNTPs, 0.5 unit Hot Start Taq DNA polymerase (Bioneer), 0.4 µM of each primer, and 10 ng template DNA. Amplification was conducted in a thermal cycler (Model PC 808, ASTEC, Fukuoka, Japan) as follows: predenaturation at 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. The amplification products were electrophoresed on 2% agarose gel stained with ethidium bromide at 150 V for 12 min.
For multiplex PCR, the optimized concentration of primers (Table 1) and 1 unit of Hot Start Taq DNA polymerase were used, and the rest of the conditions were the same as single PCR. The amplification products from the multiplex PCR were electrophoresed on 3% agarose gel stained with ethidium bromide at 150 V for 30 min. Additionally, capillary electrophoresis was conducted using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to confirm the sensitive accuracy of multiplex PCR.

DpnI, HindIII, BamHI, Phusion DNA polymerase, T5 exonuclease, Taq DNA ligase, dATP, dGTP, dCTP, dTTP, and T4 DNA ligase were obtained from New England Biolabs (UK). Taq DNA polymerase was purchased from Takara (Japan) and Hot Start Taq DNA polymerase was obtained from Bioneer (Korea). Synthesis of oligonucleotides and DNA sequencing were performed by Bioneer and Cosmogenetech (Korea). Polyclonal P9 antibody was prepared from mouse anti-serum using P9 protein as an antigen (Ab Frontier, Korea). C-terminally biotinylated endothelin-1 (bET-1) was synthesized by both of Peptron and LugenSci (Korea). Endothelin-1 (ET-1) was obtained from Sigma (USA). pGEM-T vector was purchased from Promega (USA). Phycoerythrin-conjugated streptavidin (SA-PE) and HRP-conjugated streptavidin (SA-HRP) were obtained from BD Biosciences (USA) and Thermo Scientific (USA), respectively. Trypsin-EDTA, F-12 media, FBS, Lipofectamine 2000, Fura-2-AM, and antibiotic-antimycotic solution were obtained from ThermoFisher (USA). Cell culture dishes were purchased from SPL (Korea). Ni-NTA resin was obtained from Qiagen (Germany). All other consumable reagents were purchased from Sigma (USA).

Genomic DNAs was extracted from Hanwoo longissimus dorsi muscle samples using an AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). The NCBI Primer-BLAST program was used to design primers to amplify three candidate SNPs in the GPAM 3ʹ UTR. Each primer contained a restriction site for XhoI (forward primer only) and SalI (reverse primer only). A 15-bp sequence homologous to the pmirGLO vector sequence was used for performing homologous recombination using the seamless cloning method, which has a high ligation efficiency than that of the classical cloning method. Two different SNPs in the GPAM 3ʹ UTR were amplified using PCR with primers that were designed to have 15 homologous bases with the vector. PCR was performed using HotStart Taq DNA Polymerase (Bioneer, Daejeon, Korea) by following the manufacturer’s instructions. The amplified SNPs were inserted into the XhoI- and SalI- (New England Biolabs, Beverly, Massachusetts, USA) digested pmirGLO vector (Promega, Madison, Wisconsin, USA) backbone using a Gibson Assembly Cloning Kit (New England Biolabs, Beverly, Massachusetts, USA). The presence of two different SNPs (A-A-C and G-G-T) in the 3ʹ UTR of the GPAM gene in recombinant DNA was confirmed through sequencing, and the recombinant DNA was subsequently used for luciferase assays.

The specificity of each primer set was evaluated using DNA extracted from the 19 fish species, including Atlantic cod, Pacific cod, blue whiting, haddock, and Alaska pollock. Their sensitivities were also estimated with 10-fold serially diluted DNA ranging from 10 ng to 0.01 pg.
Simplex PCR reaction was conducted using a 25 μL reaction volume mix containing 2.5 μL of the 10× buffer (Bioneer, Daejeon, Korea), 800 μM dNTPs (Bioneer), 0.5 unit of the Hot Start Taq DNA polymerase (Bioneer), 400 nM of each primer, and 10-ng template DNA. Afterward, the reaction was performed in a thermal cycler (Astec, Tokyo, Japan) under the following conditions: predenaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30s, all followed by the final extension at 72 °C for 5 min. Finally, capillary electrophoresis was used to confirm PCR products using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a DNA 1000 Lab Chip kit (Agilent Technologies).

The specificity of the multiplex PCR reaction was tested with 10 ng of genomic DNA samples isolated from the 19 fish species. The sensitivities of these samples were then measured using genomic DNA from 10 ng to 0.01 pg. Subsequently, the multiplex PCR reaction process for the simultaneous identification of Atlantic cod, Pacific cod, blue whiting, haddock, and Alaska pollock was conducted with a 25 μL reaction volume mix, containing 2.5 μL 10× buffer (Bioneer, Daejeon, Korea), 800 μM dNTPs (Bioneer), 1 unit of Hot Start Taq DNA polymerase (Bioneer), 0.4 μM Atlantic cod primers, 1 μM Pacific cod primers, 0.6 μM blue whiting primers, 0.4 μM haddock primers, 0.28 μM Alaska pollock primers, and 10 ng template DNA. Multiplex PCR condition is identical with the simplex PCR procedure as mentioned above. All PCR amplicons obtained from the multiplex PCR were confirmed via capillary electrophoresis.