The procedure was carried out according to that used by Tynkkynen et al. (1999) (link), slightly modified and described in detail in our previous paper (Brzychczy-Wloch et al., 2010 (link)). Bacterial DNA had been digested with SmaI enzyme (Thermo Fisher Scientific), and restricted fragments had been separated in a CHEF-DR III device (Bio-Rad). The genetic similarity between the isolates was calculated using Molecular Analyst software (Applied Maths). The parameters used for clustering were the Jaccard index and unweighted pair-group method with arithmetic mean (UPGMA), where tolerance was 3% and optimization was 0.5%. Genotype patterns were compared according to the guidelines of van Belkum et al. (2007) (link). Isolates with ≥80% similarity in PFGE patterns were considered to be indistinguishable and were assigned to the same subtype. The cut-off value was determined on the basis of the number and position analysis of the bands.
Smai enzyme
Manufactured by Thermo Fisher Scientific
Sourced in Canada, Lithuania
SmaI is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 5'-CCCGGG-3'. It is commonly used in molecular biology and genetic engineering applications for DNA manipulation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chef dr 3 device, by Bio-Rad (1 mentions)
Dreamtaq green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Fpquest software, by Bio-Rad (1 mentions)
Chef bacterial genomic dna plug kit, by Bio-Rad (1 mentions)
Chef dr 3 apparatus, by Bio-Rad (1 mentions)
Chef dr 2 system, by Bio-Rad (1 mentions)
Incert agarose, by Lonza (1 mentions)
4 protocols using smai enzyme
1
Bacterial DNA Fingerprinting via PFGE
2
P2Y12 Gene Polymorphism Analysis
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The 52 (G>T) (rs6809699) and 34 (C>T) (rs17602729) polymorphisms of the P2Y12 gene was analyzed with the PCR-RFLP (restriction fragment length polymorphism) method. The primer sets used were: 5′-AAT AAT AAT TCA CCT CTG CGC CCG G-3′/5′-CCG GAT TTG AAA GAA AAT CCT CA-3′ for the 52 (G>T) polymorphism, and 5′-TTT AGA GGA GGC TGT GTC CAA-3′/5′-AAT AAT GTT ACC AGG CGC AGA GGT GAA-3′ for the 34 (C>T) polymorphism. The PCR was performed with 25 μL DreamTaq Green PCR master mix (Thermo Scientific, Pittsburgh, PA, USA) with 5 μL (75 ng) DNA, 1 μm of forward and reverse primers and PCR grade water in a total reaction volume of 50 μL. An ABI PRISM™ 9700 thermal cycler (Applied Biosystems, Grand Island, NY, USA) was used for the PCR reactions. The thermal cycling conditions were: an initial denaturation step at 94 °C for 3 min. and 35 cycles at 94 °C for 20 seconds, 57 °C for 20 seconds, and 72 °C for 25 seconds; a final extension was performed at 72 °C for 3 min. An SmaI enzyme (Thermo Scientific) was used for digestion of the amplification product for the detection of the 52 (G>T) polymorphism. The PCR product used to detect the 34 (C>T) polymorphism was digested with Tsp509 I (synonime TasI) (Thermo Scientific). The products were size-fractionated on a 2.0% agarose gel.
3
Molecular Typing of S. aureus Isolates
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To demonstrate the clonal diversity of the analyzed S. aureus strains, they were subjected to molecular typing by pulsed-field gel electrophoresis (PFGE), using CHEF Bacterial Genomic DNA Plug Kits (Bio-Rad, Marnes-la-Coquette, France) according to the procedure described by Masiuk et al. [15 (link)]. Briefly, digestion of whole-genomic DNA was performed using the SmaI enzyme (MBI Fermentas, Ontario, Canada) according to the manufacturer’s protocol. PFGE was conducted using a CHEF DR III apparatus (Bio-Rad, Marnes-la-Coquette, France) in 2% agarose gel (DNA Gdansk, Gdansk, Poland) and 1×TBE (Tris-borate-EDTA) buffer with specific parameters of electrophoresis described by Masiuk et al. [15 (link)]. After electrophoresis, the gel was stained with ethidium bromide in a covered container for 30 min, visualized and photographed using a GelDoc-It2 Imager gel imaging system (Analytik Jena US LLC, Upland, CA, USA). S. aureus ATCC 25904 and S. aureus ATCC 6538 strains were included in the study.
The PFGE macrorestriction profiles were analyzed using FPQuest software (Bio-Rad, Marnes-la-Coquette, France). Classification of individual restriction patterns for each genetic profile was conducted using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and Dice’s coefficient (2.0%). PFGE results were presented as a dendrogram.
The PFGE macrorestriction profiles were analyzed using FPQuest software (Bio-Rad, Marnes-la-Coquette, France). Classification of individual restriction patterns for each genetic profile was conducted using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and Dice’s coefficient (2.0%). PFGE results were presented as a dendrogram.
4
Pulsed-Field Gel Electrophoresis of E. cecorum
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PFGE was done as previously described [26 (link), 34 (link)–36 (link)]. Genomic DNA from all isolates was embedded in 2% agarose plugs (InCert Agarose, Lonza, Rockland, USA). DNA was digested with the SmaI enzyme (20 U/μl; Fermentas, Lithuania). Electrophoresis of digested fragments was carried in 1% agarose on CHEF DRII system (Bio-Rad Laboratories, Berkeley, CA, USA) using 0.5xTBE at 14°C. The initial and final switch time was 0.5 and 35 s respectively, at 6V/min for 24 h. The DNA banding patterns were analysed with Gel Compar II BioNumerics v. 7.0 software (Applied Maths, Belgium) and cluster analysis was performed by UPGMA based on the Dice similarity coefficient, with optimization and position tolerance set at 1%. EC isolates were clustered using > 80% homology cut-off, above which strains were considered to be closely related and assigned to the same PFGE type. The reference strain E. cecorum ATCC 43198 was used as control.
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