Gel out kit

Three DNA segments encompassing the studied SNPs were amplified using polymerase chain reaction (PCR). All primers are listed in S2 Table. PCR was performed using an MJ Mini gradient PCR apparatus (Bio-Rad, Hercules, CA, USA) in 20-μl reaction mixes containing 200 ng of genomic DNA, 0.2 mM dNTPs, Taq buffer with KCl (1:10 dilution), 1.5 mM MgCl₂, 0.2 mM forward and reverse primers, and 1 unit of Taq polymerase (Fermentas, Vilnius, Lithuania). The PCR conditions are shown in S3 Table. The resulting DNA fragments were purified with a gel extraction kit (Gel-Out kit; A&A Biotechnology, Gdynia, Poland) and sequenced using the amplification primers. The genotypes (P^1NORP², P¹P², P^1NORP¹, P¹P¹, and P²P²) were assigned based on the SNP that best correlated with the P₁/P₂ blood typing and the c.631C/G status. Nucleotide differences between hetero- and homozygotes for individual P₁/P₂-related SNPs are shown in S1 Table. The pp genotype of the sole p individual in the cohort was confirmed previously [21 (link)].

Genetically, the strains were identified using ITS1 (5′-tccgtaggtgaacctgcgg-3′) and NL4 (5′-ggtccgtgtttcaagacgg-3′) primers, which cover the whole rRNA coding region including the D1/D2 domain of the large subunit of rDNA as well as the ITS (internal transcribed spacer) domain (and the intervening 5.8S rRNA gene) [62 ]. DNA was extracted from cells growing on YPD medium for 24 h using the protocols of Hoffman and Winston [63 (link)]. Phire Plant Direct PCR Master Mix was used for the PCR reaction (Thermo Scientific, Waltham, MA, USA). The samples were run in 1% agarose electrophoresis and the obtained bands were extracted from the gel using a Gel-Out kit (A&A Biotechnology, Gdańsk, Poland). The amplicons were sequenced in both directions using the primers mentioned above (Genomed S.A., Warsaw, Poland). Results obtained from sequencing were analyzed using the Basic Local Alignment Search Tool (BLASTN) available in the public domain.

Plasmid DNA was isolated using Plasmid Mini AX (A&A Biotechnology) kit following manufacturer’s instruction. Plasmids were separated in a 0.7 % agarose gel, and molecular size of the bands was determined with GelCompar II 3.5; E. coli V-517 plasmids were used as molecular size markers. All plasmids were cut off the gel and eluted by Gel-Out kit (A&A Biotechnology). Integrons in plasmids were detected as described above.

Common genetic manipulation procedures were performed according to standard protocols of Sambrook and Russell [37 ]. Plasmids of Paracoccus spp. were isolated using the method of Birnboim and Doly [45 (link)], and when required, the DNA was further purified by CsCl-ethidium bromide gradient centrifugation [37 ]. Plasmid DNA was isolated from E. coli cells using a Plasmid Mini Kit (A&A Biotechnology). Restriction endonucleases and T4 DNA ligase were used according to the supplier’s instructions (Thermo Scientific). Polymerase chain reaction (PCR) was carried out using Pfu or Phusion DNA polymerases (Thermo Scientific) following the manufacturer’s instructions. Amplifications using these thermostable DNA polymerases were performed in a Mastercycler (Eppendorf) with synthetic oligonucleotide primers and appropriate DNA templates. The PCR-amplified DNA fragments were analyzed by electrophoresis on 0.8% agarose gels and, where necessary, purified using a Gel-Out Kit (A&A Biotechnology). Chemical transformation of E. coli strains was performed according to the method of Kushner [46 ]. Triparental mating to introduce plasmids into Paracoccus spp. cells was performed as described previously [47 (link)].

Yeast strains were transformed using the LiAc/ssDNA/PEG method [160 (link)]. Total DNA was isolated from yeast cultures using the Genomic Mini AX Yeast Spin Kit (A&A Biotechnology, Gdansk, Poland). E. coli cells were transformed as described by Sambrook and Russell [161 ]. Plasmids were isolated from bacteria using the Plasmid Mini Kit (A&A Biotechnology). DNA was extracted from the agarose gel using the Gel-Out Kit (A&A Biotechnology) and purified after enzymatic reactions using the Clean-Up Kit (A&A Biotechnology). Restriction enzymes and DNA ligase (Thermo Scientific, Waltham, MA, USA) were used as recommended by the supplier.

The nested PCR was carried out for amplification of EBNA-2. The sequence of primers used for PCR was as follows: outer pair 5′ – TTT CAC CAA TAC ATG ACC C – 3′, 5′ – TGG CAA AGT GCT GAG AGC AA – 3′ and inner pair 5′ – CAA TAC ATG AAC CRG AGT CC – 3′, 5′ – AAG TGC TGA GAG CAA GGC MC – 3′. 2 μl of extracted DNA was subjected to the PCR mixture with the concentration as described above. The first-round amplification consisted of the activation of polymerase 95 °C for 15 min, 35 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min and the final extension at 72 °C for 5 min. The second-round amplification was performed with 1 μl of first round PCR product in 30 cycles with an annealing temperature at 60 °C. The amplicons 368 bp, 473 bp in length (depending on the EBV type EBV-1 and EBV-2, respectively) were separated on 2% agarose gel and purified using a Gel-Out kit (A&A Biotechnology, Poland) for further analysis. Purified PCR products were sent to Genomed Warsaw company for sequencing.

To determine the repR transcription start site a 2nd Generation 5′/3′ RACE Kit (Roche) was used according to the manufacturer’s instructions with some modifications. Since the pIGRK sequence contains a high level of A + T pairs, Poly [C] tailing of cDNA was applied instead of Poly [A] tailing. Consequently the Oligo dT-anchor primer (included in the kit) was replaced by an Oligo dG-anchor primer (oligo 32, [Additional file 4: Table S3]), during the primary PCR. For cDNA synthesis, 1 μg of total RNA was used with the gene-specific primer RACESP1 (oligo 39, [Additional file 4: Table S3]). Two separate PCR amplifications were performed using dG-tailed cDNA as the template, the first with an Oligo dG-anchor primer (included in the kit) and gene-specific primer SP2RACE (oligo 33, [Additional file 4: Table S3]). The products of these primary reactions were then used as the template in a secondary PCR, with PCR anchor primer (included in the kit) and gene-specific primer SP3RACE (oligo 34, [Additional file 4: Table S3]). The amplified DNA fragment was visualized by agarose gel electrophoresis, then purified using a Gel-Out kit (A&A Biotechnology) and sequenced.