Purelink dna mini kit

The details of the laboratory procedures such as haematological measurements, microscopy for demonstration of any asexual form of P. falciparum, DNA extraction and quantification are described in details in our previous publication [19 (link)]. In summary, haematological measurements were determined using Beckman Coulter counter ACTdiff2™, P. falciparum infection status was determined by microscopy. PureLink® DNA Mini Kit (Invitrogen life technologies, USA) was used for DNA extraction while DNA quality and quantity was assessed using Nano Drop ND-1000 spectrophotometer (Thermofisher Scientific, San Diego, CO, USA) and stored at -20 °C prior to use.

A panel of 88 DNA samples comprising positive and negative ones confirmed by microscopy (S3 Table) was obtained from biological samples collected by two sterile cervical brushes, used to scrap the patients’ lesions. The criteria to define positive or negative samples was the visualization or not of amastigote forms in skin lesion specimens, by microscopical examination. The samples were stored in RNALater solution (Ambion, Carlsbad, CA/USA) and kept at the Laboratory of Genetics Epidemiology from Fiocruz Rondônia (Fiocruz/RO), Brazil. Among the 88 samples, those in which it was possible to identify the Leishmania species, 9 were identified as L. (V.) guyanensis and the others (47) as L. (V.) braziliensis, as expected, once this are the main species causing LTA in that region. All clinical samples assays were done blindly. The DNA was extracted using the PureLink DNA MiniKit (Invitrogen, Carlsbad, CA/USA), according to manufacturer’s instructions, excluding the incubation at 55°C step. These steps were carried out in Fiocruz/RO.
Authorization from the Ethics Committee in Research of the Rondônia Tropical Medicine Center (CEP/CEPEM) was obtained under the CAAE Ethical Appraisal number 0020.0.046.000–11. Patients with clinical suspicion of ATL, attended at the hospital Cemetron/RO, were invited to participate in this study and those who accepted, signed the Informed Consent Form.

Genomic DNA (gDNA) was prepared from frozen pellets containing 10⁶ total PBMCs with the PureLink DNA Mini Kit (Invitrogen). One µg of gDNA was treated with sodium bisulfite using the EpiTect Plus DNA Bisulfite Kit (Qiagen). Real-Time PCR amplification of methylated and demethylated FOXP3i1 sequences was performed in a final volume of 25 µl with the Rotor-Gene Probe PCR Kit (Qiagen), 300 nM of each primer and 100 mM of probe in a 72-well rotor on Rotor-Gene PCR 6000 Realtime Analyser (Corbett Life Science). Two-step thermal cycling was started with a first denaturation at 95°C for 3 minutes followed by 45 cycles at 95°C for 3 seconds and 64°C for 30 seconds. Sequences of primers and probes are indicated in Table S2. The percentage of demethylated sequences is calculated as follows: 2∧(Ct methylated −Ct demethylated)/[2∧(Ct methylated −Ct demethylated)+1]*100.

Following an initial proteinase K lysing step at 50 °C for 3 h, DNA extractions were conducted with PureLink DNA mini kit (Invitrogen) according to manufacturer’s instructions. Polymerase chain reactions were conducted with partial-adapter primers 18S_0587_F (5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT CCG CGG TAA TTC CAG CTC-3′) and 18S_0964_ R (5′-G ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TGA TCC CYY AAC TTT CGT TCT TGA-3′), targeting the V4 region of the 18S ribosomal RNA gene^{40 (link)}. Amplicons were then purified with Ampure XP Purification magnetic beads and sent to GENEWIZ (South Plainfield, NJ) for sequencing. All populations were sequenced in three separate runs on Illumina HiSeq, according to the company’s automated EZ-Amplicon pipeline.

The repair plasmids were assembled as follows: SalI restriction site (RS), homology arm, GGSG linker, KpnI RS, mAID, GSG linker, T2A, NheI RS, hygromycin or neomycin resistance marker, XhoI RS homology arm and MluI RS. The mAID tag was templated within the original full-length degron according to (43 (link)) from a plasmid (pMK38) kindly provided by Prof. Masato Kanemaki. The T2A-NheI-Neo residence marker region was templated from a plasmid kindly provided by Prof. David Bentley. All additional features were introduced through respective design of PCR oligos. Templates for PCR, plasmid DNA or genomic DNA (gDNA) were purified using the GeneJET Plasmid Miniprep Kit (Thermo Fisher) or the PureLink^® DNA Mini Kit (Invitrogen), respectively. For all PCR reactions, Phusion^® High-Fidelity Polymerase (NEB) was used following the manufacturer’s instructions. Initially, a ‘scaffold’ vector was constructed using NEBuilder HiFi DNA Assembly (NEB), including restriction digest of the backbone and PCR-generated inserts with 20-nt homology overhangs on both sides. All subsequent target-specific repair templates were generated by switching of the homology arms or resistance cassettes by conventional cloning. Correct assembly was confirmed by Sanger sequencing.

Genomic DNA was extracted from the whole-blood samples using the PureLink DNA Mini Kit (Invitrogen, Hilden, Germany) as per the manufacturer’s instructions. DNA quality was assessed using a NanoDrop Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). DNA samples of 183 individuals were genotyped using the Illumina Canine 170K or 220K genotyping arrays (Neogen Inc., Lincoln, NE, USA).