Abi bigdye terminator kit

Genomic DNA was extracted from skin or liver following the standard phenol–chloroform protocol⁶⁵ . Repetitive sequences from Bufo bufo and E. calamita were revealed by digestion of genomic DNA with several restriction endonucleases (EcoRI, HindIII, PstI, BamHI, AluI, SalI, KpnI y SmaI) according to the manufacturers’ protocols and subsequent agarose gel electrophoresis. After BamHI digestion, the intense bands of about 800 bp and 1600 bp were eluted (GeneJET Gel Extraction Kit, Thermo Fisher Scientific (Waltham, Massachusetts)) and cloned into BamHI-digested pUC19 vector (Fermentas (Vilnius, Lithuania)). Competent JM109 bacteria were transformed by thermal shock. Recombinant clones were denaturalized, dot-blotted onto nylon membranes and hybridized using the BamHI-800 digoxigenin-labelled band as a probe. Positive clones were sequenced using ABI BigDye Terminator kit (Applied Biosystems (Massachusetts, USA)) and T7 or SP6 universal primers. Nucleotide sequences were obtained in an ABI PRISM 3730xl capillary DNA analyser at the DNA sequencing Service from the Centro Nacional de Investigaciones Oncológicas (CNIO (Madrid, Spain)).

Case records of patients attending the Pediatric Rheumatology Clinic, Advanced Pediatrics Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India, were reviewed. A detailed analysis of the clinical profile, investigations, treatment, and outcome of children with BS was recorded. An ophthalmological examination of these children was carried out at the uveitis clinic, Advanced Eye Centre, PGIMER, Chandigarh. The genetic diagnosis was confirmed at Pediatric Immunology Laboratory, PGIMER, Chandigarh.
Peripheral blood samples were collected for molecular analysis after obtaining informed consent. Exon-4 of NOD2 gene was amplified using polymerase chain reaction (PCR) at controlled conditions using specific oligonucleotide primers, which were obtained from the Resource of Asian Primary Immunodeficiency Database (RAPID) (15 ). The PCR products were qualitatively examined by 1.5% agarose gel electrophoresis followed by purification and direct sequencing using the ABI Big Dye Terminator kit and ABI 3500 Gene Analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing results were analysed using Codon Code Aligner software (Codon Code Corporation, Centerville, MA, USA).

Aiming at confirming the specificity of the primers, we cloned the PCR product into plasmid using the vector pGEM®-T Easy (Promega, Madison, USA) according to the manufacturer’s instructions. The cloned plasmids were purified using QIAprep Spin Miniprep kit (Qiagen, Germany) and sequenced using the T7 (foward) and SP6 (reverse) oligonucleotides with ABI BigDye Terminator Kit on an ABI 3500xl Genetic Analyzer Sequencer (Applied Biosystems, Foster City, USA). Electropherograms were analyzed manually on the BioEdit v.7.2.0 software and compared to GenBank sequences using the BLAST algorithm, confirming the primer specificity in the amplification of the 126 base pair fragments.

The extraction of An. nivipes genomic DNA in individual mosquitoes was carried out following the producer’s manual (QIAamp^® DNA Mini Kit, Germany). The amplification for approximately 620 bp of COII gene was carried out using primers, LYS-R (5′-ACTTGCTTTCAGTCATCTAATG-3′) and LEU-F (5′-TCTAATATGGCAGATTAGTGCA-3′). The overall PCR reaction volume reached 20 µl and the mixture of PCR reagent comprised 2 µl of DNA, 0.05 unit of Takara Taq (Dalian, China), 0.3 µM of the respective primer, 0.2 mM of dNTPs, and 2.5 µl of 10× Buffer (15 mM MgCl2, 100 mM Tris–HCl ^PH=8.3 as well as 500 mM KCl). Besides, the cycling parameter included 95 °C, 5 min; 95 °C/1 min, 51 °C/1 min, 72 °C/2 min for 35 cycles; with a final extension of 72 °C for 10 min. The PCR products were analysed by 1.5% agarose gel electrophoresis stained with GoldView (Solarbio, China), under UV transillumination. The sequencing reaction proceeded in both directions using an ABI Big Dye Terminator Kit (Applied Biosystems, Thermo Fisher Scientific) and was analysed through ABI Prism 3500xL Genetic Analysis Tool (Applied Biosystems, Thermo Fisher Scientific) in Shanghai (Sangon Biotech).

Dotblot and aDNA hybridization procedures were conducted as described elsewhere [17 (link)]. Human DNA was used as negative control. DNA probes of 93 and 113 bp, corresponding to MTC molecular targets IS6110 and IS1081, respectively [35 (link),36 (link)], were prepared by PCR. DNA from four MTC type strains were used as hybridization positive controls for IS6110 and IS1081 probes: M. tuberculosis T92 (ATCC27294^T), M. tuberculosis H37Rv (ATCC27294^T), M. bovis BCG (ATCC19210^T) and M. africanum T85 (ATCC25420^T). The pPCRs were purified by a GFX PCR DNA and Gel Band Purification kit (GE HealthCare) and directly sequenced using an ABI BigDye Terminator kit (Applied Biosystems) according to the manufacturer’s protocol, with analysis in both directions, on an ABI 3730 (Applied Biosystems) automated sequencer. BioEdit v. 7.0.4 (Department of Microbiology, North Carolina State University, USA), and Lasergene Seqman v. 7.0.0 (DNASTAR, Madison, WI, USA) were used for editing and sequence analysis. Sequencing and sequence analysis were performed to confirm MTC molecular targets. Probes were labeled by chemiluminescence using Gene Images Alkphos Direct Labeling and Detection Systems (Amersham) as described elsewhere [17 (link)].