Haeiii

Genomic DNA was extracted from pellets of the Leishmania cultures using High Pure PCR Template Preparation Kit (Roche Diagnostics, Germany) according to the manufacturer′s instructions. Samples were stored at −20 °C until use. DNA samples from Iranian reference strains of L. tropica (MHOM/IR/02/Mash10/Accession No. EF653267), L. major (MRHO/IR/11/GOL-2/ Accession No. JN860745) and L. infantum (MCAN/IR/ 07/Mash-ir1/ Accession No. EU810776) were used as positive controls. The DNA samples were assessed for the Leishmania-specific ribosomal internal transcribed spacer 1 region (ITS1) by PCR amplification using primer pairs of LITSR (F: 5′-CTGGATCATTTTCCGATG-3′) and L5.8S (R: 5′-TGATACCACTTAT CGCACTT-3′). Amplification was carried out using PCR-Ready Premix (Roche, Germany) in a 25μl reaction. The amplification conditions included those described previously (20 (link)). PCR products (8μl) were digested with the restriction endonuclease enzyme HaeIII (BsuRI) (Fermentas, Germany) for the species identification according to the manufacturer′s instructions. Amplicons of nearly 300–350bp and restriction fragments were analyzed using 1.5–3% agarose gels containing safe stain, visualized under UV and compared with those from reference strains of L. tropica, L. major and L. infantum.

Two restriction enzymes, namely, HaeIII (Fermentas, Lithuania) and RsaI (Thermo Scientific), were used to digest the amplified DNA fragments as per the manufacturer's protocol. The digestion products were separated using electrophoresis on a 2% agarose gel (Sigma) in 0.5x TAE buffer and analyzed under UV light.

For the extraction of DNA a loopful of mycobacteria grown on 7H10 medium was suspended in 300 mL of TE buffer (10 mMTris, 1 mM EDTA pH 8.5) and subjected to 3 cycles of boiling (10 min. at 100°C) and freezing (20 minutes at -20°C). The PRA technique (PCR-Restriction Enzyme Analysis) was used to identify the mycobacterial isolates. A 439 bp fragment was amplified from the hsp65 gene for PCR. The DNA sequence of the PCR product was then digesting with the enzymes BstEII (Fermentas, Glen Burnie, MD, USA) and HaeIII (Fermentas, Glen Burnie, MD, USA) according to [21 ]. The products of the digestion reaction was separated and visualized by gel electrophoresis. The pattern of bands obtained was then compared with the banding patterns obtained from reference strains according to the database PRASITE (http://app.chuv.ch/prasite).

Total DNA was extracted by GUTC (Guanidinium-Tris-CDTA buffer with celite) method (Terefework et al., 2001 (link)) from purified bacteria. 16S rDNA and intergenic spacer region (IGS) of the strains were amplified for restriction fragment length polymorphism analysis. Primer pairs P1, P6 and pHr(F), p23SR01(R) (Table 1) were used for polymerase chain reaction (PCR) amplification. Amplification products (5 μl) were digested separately by four restriction enzymes HinfI, TaqI, MspI, and HaeIII following the manufacturer's instructions (Fermentas, EU). The fragments were separated by gel electrophoreses in 2% agarose with 0.5 μg ml⁻¹ ethidium bromide at 80 V for 3 h and photographed. Amplified ribosomal DNA restriction analysis (ARDRA) and IGS-RFLP were done by combining the results from the four restrictions. Clustered analysis of combined ARDRA and IGS-RFLP (CACAI) was conducted by UPGM clustering algorithm in the NTSYS program (Rohlf, 1990 ).

The amplified DNA was then digested using four endonuclease restriction enzymes AluI, HaeIII, HhaI and TaqI (Fermentas) separately, in order to obtain RFLP fingerprinting.
The amplified 16S rRNA-encoding gene products were digested following the manufacturer’s instructions. DNA fragments were separated by electrophoresis on 1.5% w/v agarose gel 1X TAE stained with SYBR® Safe (Invitrogen) for 1 h at 100 V, 400 mA. The reference used was PerfectSize DNA molecular weight 100 bp XL Ladder (5 Prime). Scanned images of the gels containing DNA-RFLP were captured with Quantity One software (Bio-Rad).

Five endonucleases, TaqI, BamHI, HindIII, HaeIII, MspI (Fermentas GmbH, St. Leon-Rot, Germany) were used for RFLP analysis of ITS region of P. syringae. pv. tomato strains with the primer pair 1406-f and 23 S-r (Fisher and Triplett 1999) and four endonucleases (TaqI, HaeIII, HindIII and NeaI) were used for digestion of specific fragment from the hrpZpst gene. Amplified DNA products were precipitated in 2.5 µl ethanol (96%) and 0.1 µl of NaAc (3 M) with the final pH 3.2. Products were further deliquesced in 11 µl of Tris acetate (TAE) buffer containing EDTA (1 mM), 10 mM TRIS with the final pH 8.0. Then, the DNA solution (1 µl) was analyzed to estimate DNA concentration in agarose gel (2%) electrophoresis. For restriction analysis with each selected enzyme, 2 µl DNA was used and RFLP products were visualized in 2% agarose gel.