Klenow polymerase

In order to obtain the complete genome sequences of samples identified as CoV-positive in our earlier studies (166 positives including 140 gamma- and 26 deltacoronaviruses), selected samples with the highest viral load confirmed by PCR were subjected to Next Generation Sequencing (NGS) using the MiSeq Personal Sequencer platform (Illumina, USA) offered by the Department of Omics Analysis of PIWet-PIB or the commercial service Genomed SA. (Warsaw, Poland). A total of 27 coronavirus-positive field specimens were subjected to NGS attempts. Briefly, samples (cloacal swabs) were treated with TURBO DNase (Life Technologies, USA) and RNase One (Promega, USA) to remove DNA and extracapsid RNA. The isolation of viral RNA from such treated samples was carried out and then retrotranscribed into DNA using a Superscript IV First-Strand cDNA Synthesis Kit (Invitrogen, USA) and the second strand was synthesized with the addition of Klenow polymerase (New England Biolabs, USA). A 300 bp long paired-end DNA library was prepared using a Nextera XT sample preparation kit (Illumina Inc) and sequencing was performed using a MiSeq Reagent kit v3 (Illumina Inc).

DNA manipulations were carried out using standard molecular techniques [17] . Restriction enzymes, calf intestinal alkaline phosphatase (CIP), T4 polynucleotide kinase, and Klenow polymerase were obtained from New England Biolabs, Pfu polymerase from Stratagene, AmpliTaq polymerase from Applied Biosystems, and primers from Sigma Genosys. Plasmids used or created in this study are listed in Table 2, while primers are listed in Tables 3 and 4. Genomic DNA was isolated and PCRs for brp gene linkage analysis were completed as described [7] (link), [8] (link). For Southern blotting, fragments specific for the brpC or brpI genes were generated via PCR with primer pairs RUG17/RUG18, and CAP27/CAP28, respectively. Production of radiolabeled probes and hybridizations were performed as described [7] (link) using ca. 10⁸ cpm/ml of probe per hybridization.