High fidelity dna polymerase

A reference E. coli ATCC 25922 strain was used to prepare a bacterial suspension. A few colonies of one-day E. coli bacteria were mixed with 700 µL nuclease-free water, then the suspension was vortexed and centrifuged for 2–4 s (2700 rpm). Bacterial cell wall structures were disrupted mechanically, and high-fidelity DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania) was used for the gene amplification; no further DNA purification was required.

For construction of mutant viruses by BAC mutagenesis, a two-step lambda red-mediated homologous recombination was performed in Escherichia coli as previously described [17 (link)]. In brief, PCR using specific primers with homologous sequences was conducted with high fidelity DNA polymerase (ThermoFisher, Waltham, MA, USA). PCR product was purified and electroporated (1800 V, 100 Ω, 2.5 μF) into the BAC-containing competent cells. Then the competent cells were spread onto Luria–Bertani (LB) agar plate containing kanamycin (30 μg/mL), ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL) until the bacterial colonies are observed. The triple antibiotic-resistant colonies were picked up and inoculated into fresh LB cultural media. In the recombination step, the final 0.2% arabinose was added to the growth medium resulting in induction of enzyme expression and cleavage.

OLE1 encoding ∆ (9) FA desaturase (GenBank Accession Number: NC_001139.9) was amplified from genomic DNA of S. cerevisiae CEN.PK 113-7D using high-fidelity DNA polymerase (Thermo Fisher Scientific) with the primers OLEF (5′-CGCGGATCCATGCCAACTTCTGGAACTACTAT-3′) and OLER (5′-CCCAAGCTT TTAAAAGAACTTACCAGTTTCGTA-3′). A 1533-bp PCR fragment including the entire coding region was obtained and then inserted into the 2-micron plasmid p426TEF [62 (link)] under the TEF promoter with BamHI/HindIII to yield the plasmid p426TEF-OLE1.
The expression levels of ∆ (9) FA desaturase and squalene epoxidase (ERG1) were knocked down using the antisense oligonucleotide method, as described previously [63 (link)]. The antisense oligonucleotide of the conserved catalytic domain of OLE1 was created by annealing the primer pair ELOF (5′-CCCAAGCTTTGGGGCCACTCTCACAGAATTCACC ATCGTTAC-3′)/ELOR (5′-CGCGGATCCGTAACGATGGTGAATTCTGTGAGAGTGGCCCCA-3′). The antisense oligonucleotide of the conserved catalytic domain of ERG1 was created similarly using the annealing primer pair ELOF (5′-CCCAAGCTTCGATTGTGTCAACAAACCCGTTGAATTTCTGTC-3′)/ELOR (5′-CGCGGATCCGACAGAAATTCAACGGGTTTGTTGACACAATCG-3′). The two 33-bp antisense DNA fragments, thus, obtained were then inserted into the plasmid p426TEF under the TEF promoter with BamHI/HindIII, separately, forming the plasmids p426TEF-ROLE1 and p426TEF-RERG1.

According to the de novo transcriptome assembly of S. japonicum from GenBank with the project accession number of PRJNA343582 [23 (link)], we obtained information about complete sequence of the SjTK4 gene. Primers were designed and synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The forward and reverse primers were 5′-ATG AAT GTG ACT AAT AAT GTG GTA GTA ACT CCA-3′ and 5′-TTA AAA TGA TAT TCC ATC ACT ACC ACC A-3′, respectively. For PCR amplification, a 50 μl total reaction volume was prepared containing 0.2 μg cDNA, 5 μmol of each primer, 0.5 mmol dNTPs and 2.5 U high-fidelity DNA polymerase (Thermo Scientific). After a denaturation step at 94 °C for 5 min, thermal cycling was performed as follows: 94 °C for 45 s, 60 °C for 1 min, 72 °C for 1 min for 30 cycles and a final extension at 72 °C for 8 min. The PCR products were purified by agarose gel electrophoresis and sent for sequencing to Sangon Biotech Co. Ltd. (Shanghai, China). The sequencing result was uploaded to the National Center for Biotechnology Information (NCBI) database (GenBank accession number: KX984125).