Bigdye terminator mix

PCR amplifications of positive to MMTV-like sequences were performed again using the same mixture and thermocycler conditions but replacing fluorescent primers with non-fluorescent ones. PCR products were separated by 2% agarose gel electrophoresis, and gels were stained with Invitrogen SYBR Safe DNA Gel Stain 0.05 (Life Technologies Ι Carlsbad, CA, USA) and then visualized by a UV illuminator. The bands from sample that was positive to MMTV env-like were cut and sequenced after cleaning up with the MinEluite Gel Extraction Kit (Qiagen, Venlo, Netherlands) using Big Dye Terminator mix (Applied BioSystems, Warrington, UK). Sequencing reactions were run on a 3500 Genetic Analyzer (Applied BioSystems, Foster City, CA, USA).

Genomic DNA was isolated from peripheral blood leucocytes using the QIAamp Blood Mini Kit. The HbS confirmation was carried out using Covalent Reverse Dot Blot technique¹² . 3 polymorphisms in the HMOX1 gene based on their presumptive functional effects and on the basis of the literature survey were selected. The rs3074372 (GT)n repeats were detected using fluorescently labelled primers with primer sequence as followed:F’FAM:5′AGAGCCTGCAGCTTCT3′ and R:5′ACAAAG TCTGGCCATAGGAC 3′ and PCR was conducted^{13 (link)}. The PCR products were loaded along with a size standard LIZ (− 250) (Applied Biosystems) onto ABI genetic analyser 3130 XL, under fragment analysis protocol and the fragment size was analysed by GeneMapper v4.1 (Applied Biosystems) software. The rs2071746 (A→T) polymorphism was detected by direct DNA sequencing, using BigDye terminator mix (Applied Biosystems) with already described primers and protocol^{14 (link)}. The rs2071749 (A→G) polymorphism was detected by using MluCI restriction enzyme digestion (New England BioLabs)^{13 (link)}.

PCR products were sequenced by the dideoxynucleotide method [21 ] in an automated capillary sequencer (ABI 3730, Applied Biosystems^®, USA), using Big Dye Terminator^® Mix (Applied Biosystems, EUA) with analyses performed in version 5.2 of Applied Biosystems Sequencing Analysis. Sequencing was performed in triplicate in both directions. Sequences were compared by BLAST^® (https://blast.ncbi.nlm.nih.gov) and were aligned by Clustal W with sequences of matrix protein gene available in the GenBank [22 (link)]. A phylogenetic tree was constructed using the Neighbor-joining method [23 (link)] and the evolutionary distance model Kimura 2 and bootstrap with 1,000 repetitions using MEGA 7.0 software, including an external group (MH190827.1 Mammalian 1 Orthobornavirus) [24 (link), 25 (link)].

Sample that was positive to MMTV env-like was sequenced after clean up with the QIAquick PCR Purification Kit (Qiagen, Venlo, Netherlands) using Big Dye Terminator mix (Applied BioSystems, Warrington, UK). Sequencing reactions were run on an ABI3130 XL (Applied BioSystems, Warrington, UK).

spa and MLST amplicons were purified using the DNA Clean and Concentrator kit (Zymo Research, Irvine, California, USA) following the manufacturer’s instructions with a final elution volume of 30 µl. Both forward and reverse strands were sequenced. Each reaction contained 4 µl of sterile distilled water, 2 µl of 5X Big Dye buffer (Applied Biosystems, Foster City, California, USA), 1 µl (4 µM) of the PCR primer, 1 µl of Big Dye terminator mix (Applied Biosystems, Foster City, California, USA) and 4 µl of amplicon DNA. Cycle sequencing was performed on an ABI 9700 thermocycler with cycling conditions set as: 94 °C for 5 min followed by 30 cycles of 94 °C for 15 s, 55 °C for 30 s and 68 °C for 2.5 min with a final extension of 68 °C for 3 min. Sequencing fragments were purified using Sephadex G50 resin (Sigma-Aldrich, St Louis, Missouri, USA) before loading on the Applied Biosystems 3500 Genetic Analyzer.

Plasmids or PCR fragments of genomic regions were used in a Sanger sequencing reaction with Big Dye terminator mix (Applied biosystems, Waltham, MA) and the resulting mixture was purified by ethanol precipitation. Samples were run on an ABI 3130 genetic analyzer (Applied biosystems) and analyzed using Sequencer 4.8 software (GeneCodes Inc, Ann Arbor, MI).