High fidelity dna polymerase

The full-length infectious cDNA clones (ICs) of the NDV heat-resistant strain HR09 and thermolabile strain La Sota, named cHR and cLa, respectively, were generated in plasmid TVT-7R (0.0) (Johnson et al., 2000 (link)) from six fragments that were amplified by PCR using high-fidelity DNA polymerase (New England BioLabs, United States) as described in our previous research (Ruan et al., 2020 (link)).
Three chimeric ICs, cHR-La-F, cHR-La-HN, and cHR-La-F/HN, were constructed by replacing the open reading frames (ORFs) of the F and/or HN genes in the cHR strain with the counterpart fragments from the La Sota strain (Figure 1). Briefly, the ORFs of the F and/or HN genes were amplified by PCR and then inserted into the corresponding sites in full-length ICs of La Sota according to the HiFi DNA assembly cloning method (New England BioLabs, United States). Similarly, ICs of the chimeric viruses cLa-HR-F, cLa-HR-HN and cLa-HR-F/HN were generated by replacing the ORFs of the F and/or HN genes in cLa with the corresponding fragments from cHR (Figure 1).

The potential off-target regions were predicted using the online tool, http://crispr.mit.edu/. The regions with score >0.5 and mismatch ⩽4 were considered as the potential off-target sites. They were amplified using High-Fidelity DNA Polymerase (New England Biolabs) and sequenced.

Bacterial DNA was extracted using the QIAamp DNA stool Mini Kit (Qiagen, Germany) from rat feces. DNA samples were diluted (1 ng/μl) and amplified using common PCR primers 515F/806R (the V4 variable region of the 16S rRNA), high-fidelity DNA polymerase (New England Biolabs, USA), and Phusion^® High-Fidelity PCR Master Mix kit (New England Biolabs). The PCR products were purified, pooled, and used for the construction of DNA libraries using the TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, USA). 16 s RNA sequencing was processed on the Illumina MiSeq platform (Illumina, San Diego, USA; 2 × 250 bp PE).

The M. bovis strain RM16 was transformed with plasmid pOGch expressing the red fluorescent marker mCherry. In pOGch, the mCherry coding sequence was cloned downstream of the gentamicin resistance gene aacA-aphD to produce a fusion protein (Gch). The mCherry coding sequence was obtained from Martínez-Torró et al. (73 (link)). The primers used for mCherry amplification from the plasmid pCatcherry and the gentamicin marker was amplified with its promoter region from the plasmid pMT85 are available in Table S2 (74 (link)). These two regions were assembled by overlap extension PCR using GmF_EcoRI and CherryR_BglII primers. PCRs were performed using the New England Biolabs high-fidelity DNA polymerase. The Gch PCR product was cloned into pGEM-T Easy (Promega) before subcloning at the NotI site of p20-1miniO/T plasmid (75 (link)), from which the tet gene has been extracted by PstI restriction, to generate pOGch. Plasmid constructions were verified by DNA sequencing. Transformation of pOGch in M. bovis RM16 strain was done as previously described (76 (link)). Gentamicin-selected transformants were stored at −80°C. M. bovis mCherry stock cultures were produced in SP4 medium supplemented with gentamicin (50 μg·mL⁻¹), and bacterial titers were determined by counting CFU as the wild-type strain.

To the mixture from the previous step (12 µl), 10 μl of 5× Q5 buffer (High-Fidelity DNA Polymerase, New England Biolabs), 2 μl of deoxynucleotide triphosphates (dNTPs) (10 mM each), 2 μl of i5 barcoded Nextera primer (5 μM), 1 μl of i7 barcoded Nextera primer (5 μM), 0.5 μl of Q5 Hi-Fi polymerase, and 39.5 μl of water were added and thoroughly mixed. Afterward, the mixture underwent the following steps in a thermal cycler: one cycle at 72 °C for 4 min, one cycle at 98 °C for 30 s, 14 cycles of denaturation at 98 °C for 15 s, annealing at 67 °C for 20 s, extension at 72 °C for 90 s, and cooling to 4 °C.

To determine the traQ gene sequneces of Anc(C) and M54(C), the traQ region in 10 colonies each of Anc(C) and M54(C) was amplified by PCR with the primers F_4f_2 and F_4r_2 and Phusion®; High-Fidelity DNA polymerase (New England BioLabs), and PCR products were directly sequenced by the dideoxynucleotide chain termination sequencing method (Sanger et al., 1977 (link)).

DNA samples for high-throughput sequencing were amplified using Phusion. High-Fidelity DNA polymerase (New England BioLabs) and 18 PCR cycles (for specific primers of each experiment see primers list). Libraries were prepared as Blecher-Gonen et al.53 (link). High-throughput Sequencing was performed at the G-INCPM unit at the Weizmann Institute of Science with the Illumina HiSeq 2500 platform for 2 × 125 paired-end reads.
DNA samples for Sanger sequencing were amplified using REDTaq (SIGMA-ALDRICH) with 35 PCR cycles (for primers see Supplementary Table 1). Then cleaned with Exonuclease I and Shrimp Alkaline Phosphatase (rSAP) (New England BioLabs). Sequencing was performed at the Biological services unit at the Weizmann Institute of Science with Applied Biosystems 3730 DNA Analyzer.