Dntp mix

Total RNA was isolated from sections of fresh-frozen malignant (tumor cell amount ≥70%) and matched non-malignant (tumor cell amount ≤10%) tissue samples using the Invisorb Spin Tissue RNA Mini Kit (Stratec Molecular, Berlin, Germany) according to the manufacturer's instructions. RNA quality and quantity was determined with the Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany). Up to 500 ng total RNA were reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (200 U; Life Technologies, Darmstadt, Germany), dNTP mix (10 pmol of each dNTP; GE Healthcare, Freiburg, Germany) and random hexamer primers (200 ng, GE Healthcare) according to manufacturers' recommendations.

The mangrove metagenomic DNA was used as template for ARHD gene PCR‐based amplification using the degenerate primers: ARHDf (TTY RYI TGY AII TAY CAY GGI TGG G) and ARHDr (AAI TKY TCI GCI GSI RMY TTC CA) (Bellicanta, 2005). These primers were designed to flank a highly conserved region of the alpha subunit from ARHD gene, with an expected amplicon size ranging between 300 and 329 bp. The PCR reaction was prepared to a final volume of 50 μl containing 1X Taq buffer (Invitrogen), 1.5 mmol/L MgCl₂, 0.2 mmol/L dNTP mix (GE Healthcare), 1.2 μmol/L of each primer, 1 U Platinum Taq DNA polymerase (Invitrogen) and 2 μl (12 ng/μl) of eDNA. The reaction was conducted in an Eppendorf Mastercycler Gradient (Eppendorf Scientific, New York, USA) and the program consisted of an initial denaturation at 97°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min, and a final extension step at 72°C for 5 min. PCR products were separated on 1% agarose gel (Fisher Scientific, MA) in 1X TAE buffer, using the molecular weight marker 1 Kb Plus DNA Ladder (Invitrogen) for size estimation and observed under UV light using a ImageQuant LAS 4000 system (GE Healthcare Life Sciences).

Repetitive element palindromic (rep-PCR) using the (GTG)₅ primer (5´-GTG GTG GTG GTG GTG-3´) was performed [20 (link)] with a few modifications. Briefly, 1 μL of DNA was pipetted into 24 μL of a PCR mixture containing 1 µL of the (GTG)₅ primer (50 pmol/µL), 0.5 μL of dNTP mix (10 nmol/µL of each dNTP; GE Healthcare), 0.5 µL of DNA polymerase (2 U/µL; Dynazyme II; Thermo Scientific), 2.5 μL of 10× PCR buffer (100 nmol/µL Tris-HCl, 15 nmol/µL MgCl₂, 150 nmol/µL KCl, 0.1% Triton X-100; pH 8.8, Thermo Scientific), and 19.5 µL sterile distilled water. The cycling program of the Eppendorf Mastercycler (Eppendorf, AG) consisted of an initial denaturation step at 94 °C for 7 min, 30 cycles of denaturation at 90 °C for 30 sec, annealing at 40 °C for 1 min, extension at 65 °C for 8 min and a final extension at 65 °C for 16 min. Obtained PCR products were separated on a 2% agarose gel and stained with GelRed Nucleic Acid Gel Stain.
The cluster analysis of the rep-PCR profiles was performed on similarity matrices, which were produced using the Dice’s coefficient [21 (link)] and subjected to the unweighted pair group method with arithmetic mean (UPGMA) clustering algorithm using the BioNumerics software version 7.6.1 (Applied-Maths, Saint-Martens-Latem, Belgium). A tolerance level of 1% and an optimization of 0.5% were chosen for creating the dendrogram.