DNA was isolated from peripheral blood by use of MasterPure™ DNA Purification Kit for Blood Version II (Epicentre Biotechnologies, Madison, WI, USA). Following primers pair, covering bovine THRSP exon 1 was designed using Primer3 software [13 (link)]: F 5’-GCTGTGTTGACCTACTGGC-3’, R 5’-CGGCCACCATTACCTTTCCT3’. Primers were designed based on the ENSBTAG00000011666 sequence [10 (link)]. PCR cycling was as follows: initial denaturation at 94 °C/5 min, 35 cycles of 94 °C/30 s, 61 °C/45 s, 72 °C/30 s, and final extension at 72 °C/5 min. PCR was performed in a total volume of 15 µL that contains 50–80 ng of genomic DNA, 1.5 mM MgCl2, 0.3 mM of dNTP mix, 12 pmol of each primer, and 0.35 U of Taq polymerase (EURx, Gdansk, Poland). The presence of specific amplicons (598 bp) was confirmed in 1.5% agarose gel with Perfect™ 100–1000 bp DNA Ladder (EURx, Gdansk, Poland). Sequencing of amplicons was performed by an external service (Genomed, Warsaw, Poland). PCR-RFLP method was applied to determine detected THRSP gene variants (rs42714482) [14 ]. The same pair of primers and conditions as mentioned above were used in PCR. A total of 10 µL of obtained amplicons were digested by BstC8I enzyme (SibEnzyme, Novosibirsk, Russia) in 55 °C at least 3 h. Restriction fragments were separation in 4% agarose gels with a 50 bp DNA Ladder (Genoplast, Rokocin, Poland).
Taq polymerase
Manufactured by EURx
Sourced in Poland
Taq polymerase is a thermostable DNA-dependent DNA polymerase enzyme isolated from the thermophilic bacterium Thermus aquaticus. It is a widely used enzyme in polymerase chain reaction (PCR) techniques for DNA amplification and analysis.
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Lab products found in correlation
Masterpure dna purification kit for blood version 2, by Illumina (1 mentions)
Nuclease free water, by AppliChem (1 mentions)
Mini sub cell gt system, by Bio-Rad (1 mentions)
Biometra tpersonal thermocycler, by Analytik Jena (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Applied biosystems 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Micro bio spin p 30 columns, by Bio-Rad (1 mentions)
5 protocols using taq polymerase
1
Bovine THRSP Gene Variant Analysis
2
LDHA Gene Microsatellite Polymorphism Analysis
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The (TTTAT)3-5 microsatellite polymorphism within intron 6 of the LDHA gene was analysed using conventional PCR reaction. PCR primers (Table 1 https://primer3.ut.ee/ ). PCR reaction mixtures contained ~80 ng of genomic DNA, 15 pmol of each primer, 1 x PCR buffer, 1.5 mM MgCl2, 200 μM dNTP, 0.4 unit of Taq polymerase (Eurx, Gdańsk, Poland) and nuclease-free water (AppliChem, Darmstadt, German) in a total volume of 15 μl. The PCR amplification program was as follows: 5 min initial denaturation at 94°C followed by 35 cycles (denaturation at 94°C for 30 s, primer annealing at 61°C for 30 s and product synthesis at 72°C for 30 s) and final elongation at 72°C for 5 min (Biometra T-Personal thermocycler, Biometra GmbH, Göttingen Germany). Finally, PCR products were separated by horizontal electrophoresis (80 V, 400 mA, 240 min; Mini-Sub Cell GT Systems, Bio-Rad, Hercules, CA, United States) on a 5% agarose gel (Norgen Biotek Corp., Canada) [4 ].
3
Genomic DNA Extraction and Molecular Identification
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Total genomic DNA was extracted from six worms (two from M. glareolus, two from T. troglodytes, and two from E. rubecula) following the manufacturer’s protocol (DNeasy Blood and Tissue Kit, Qiagen, Germany). The 28S rDNA locus was amplified using primers: forward—dig12 (5′-AAG CAT ATC ACT AAG CGG-3′) and reverse—1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Tkach et al. 2003 (link)). The ITS1-5.8S-ITS2 region was amplified with the following primers: NLF/NLR (5′-TTTGyACACACCGCCCGTCG-3′/5′-ATATGCTTAArTTCAGCGGGT-3′) (Van der Auwera et al. 1994 (link)). PCR reactions were performed in a total volume of 25 μl containing 3 μl of genomic DNA, 10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 150 pmol of each primer, and 2 units of Taq polymerase (EurX, Poland). The thermocycling profile was as follows: 95 °C/3 min—initial denaturation; 94 °C/30 s, 52 °C/30 s (28SrDNA) or 48 °C/30 s (ITS complex), 72 °C/90 s—40 cycles; 72 °C/7 min—final extension.
The amplification products were purified using QIAquick PCR purification Kit (Qiagen, Germany) and sequenced in both directions (Genomed S.A., Poland). The obtained sequences were deposited in GenBank under accession numbers KP682451 and KP682452.
The amplification products were purified using QIAquick PCR purification Kit (Qiagen, Germany) and sequenced in both directions (Genomed S.A., Poland). The obtained sequences were deposited in GenBank under accession numbers KP682451 and KP682452.
4
DNA Amplification and Polymorphism Analysis
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The primer sequences for the DNA amplification reactions were designed based on information reported by Zhou et al. 11 (link) and confirmed using the National Center for Biotechnology Information (NCBI) database (Table 2). The 5' end of the sense primer was labeled with TAMRA (Table 2).
Polymerase chain reaction (PCR) was carried out in a total volume of 10 μL. The reaction mixture contained 30 μg of genomic DNA, 0.1 μM each of sense primer (forward) (Metabion International AG, Planegg, Germany) and antisense primer (reverse) (Genomed S.A., Warszawa, Poland), 1 U Taq polymerase (EURX, Gdańsk, Poland), 1× PCR buffer (containing 15 mM MgCl 2 ) (EURX), and buffered dideoxynucleotide mixture (dNTP) containing 200 μM of each dNTP (Invitrogen, Life Technologies/Thermo Fisher Scientific, Foster City, USA). Amplifications were performed in a T100 ™ Thermal Cycler (Bio-Rad, Hercules, USA). The PCR cycling conditions were as follows: 95°C for 300 s, followed by 34 cycles of 94°C for 18 s, 64°C for 18 s, 72°C for 18 s, and 72°C for 600 s. Qualitative analysis of PCR products was based on electrophoresis in 2% agarose gel, visualized under ultraviolet light (UV).
Polymorphism analysis was performed using capillary electrophoresis using an Applied Biosystems ® 3130 Genetic Analyzer, equipped with 3130 Series Data Collection Software v. 4 (Life Technologies/Thermo Fisher Scientific).
Polymerase chain reaction (PCR) was carried out in a total volume of 10 μL. The reaction mixture contained 30 μg of genomic DNA, 0.1 μM each of sense primer (forward) (Metabion International AG, Planegg, Germany) and antisense primer (reverse) (Genomed S.A., Warszawa, Poland), 1 U Taq polymerase (EURX, Gdańsk, Poland), 1× PCR buffer (containing 15 mM MgCl 2 ) (EURX), and buffered dideoxynucleotide mixture (dNTP) containing 200 μM of each dNTP (Invitrogen, Life Technologies/Thermo Fisher Scientific, Foster City, USA). Amplifications were performed in a T100 ™ Thermal Cycler (Bio-Rad, Hercules, USA). The PCR cycling conditions were as follows: 95°C for 300 s, followed by 34 cycles of 94°C for 18 s, 64°C for 18 s, 72°C for 18 s, and 72°C for 600 s. Qualitative analysis of PCR products was based on electrophoresis in 2% agarose gel, visualized under ultraviolet light (UV).
Polymorphism analysis was performed using capillary electrophoresis using an Applied Biosystems ® 3130 Genetic Analyzer, equipped with 3130 Series Data Collection Software v. 4 (Life Technologies/Thermo Fisher Scientific).
5
DNA Extraction and 16S rDNA Sequencing
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To obtain DNA for taxonomic analysis, cell material was scraped from agar plates and lysed using Genejet (Thermo Fisher) solutions with addition of 10 mg/mL lysozyme instead of RNase in the first step. Upon cell lysis, samples were centrifuged at high speed for 10 min, and DNA in the supernatants was precipitated with isopropanol. Washed and dried DNA pellets were taken up in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) buffer containing 50 μg/mL RNase A and incubated for 30 min at 37 • C. The samples were extracted with chloroform, DNA was precipitated with ethanol and finally taken up in 25 μL TE buffer. Oligonucleotides 16S 339 F: 5 -CTCCTACGGGAGGCAGCAG-3 and 16S 1087 R: 5 -CTCGTTGCGGGACTTAACCC-3 for partial 16S rDNA PCR amplificationpt were designed, yielding amplicons of ∼0.75 kb that include variable regions V3-V5 of 16S rDNA (Weisburg et al. 1991) . Colony PCR was performed with Taq polymerase (EURx) using a standard PCR program. PCR fragments were cleaned up using Micro Bio-Spin P-30 columns (Bio-Rad) and sequenced (STAB-Vida). Taxonomic and phylogenetic DNA sequence analysis was performed using NCBI-Blast and MEGA X (Kumar et al. 2018) software.
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