Hinfi

In case of a CT positive result, the correspondent remaining eluate was recovered from Versant PCR plate and used for CT molecular genotyping [12 (link)]. Molecular genotyping was performed by omp1 gene semi-nested PCR followed by RFLP analysis, as previously described [13 (link), 14 (link)]. Briefly, the first product of 1033 base pairs (bp) was amplified using the following paired primers: SERO1A (5’-ATGAAAAAACTCTGAAATCGG-3’) and SERO2A (5’-TTTCTAGATCTTCATTCTTGTT-3’). Then, 1 μL of the first-round PCR product was used to amplify a 978 bp fragment, using the following primers: SERO2A and PCTM3 (5’-TCCTTGCAAGCTCTGCCTGTGGGGAATCCT-3’). After the PCR step, the amplified product was digested with AluI, DdeI and/or HinfI as restriction enzymes (Promega, Madison, USA) and visualized after electrophoresis run in ethidium bromide stained 12% polyacrylamide gel. CT serovar identification was achieved by the analysis of the specific restriction pattern.

The phage genetic material was digested with restriction enzymes in order to determine its size. The enzymes used were EcoRI, EcoRV, HinfI, and HindIII, all of which were from Promega (Madison, WI, United States). Restricted DNA was electrophoresed on 0.7% (w/v) agarose gel in TAE buffer 1× (40 mM Tris-acetate, 1 mM EDTA) and visualized by UV photography after staining with ethidium bromide 5 μg/mL. The Lambda phage digested with HindIII enzyme was used as a marker.

The TL of 33 MGUS and 34 MM patients were determined by terminal restriction fragments (TRF) assay. Briefly, genomic DNA was purified using the standard method, with proteinase K treatment and phenol/chloroform extraction, from BM samples of patients. DNA (10μg) was double digested overnight by HinfI and RsaI (Promega), fractioned on a 0.8% agarose gel and transferred to a nylon membrane by Southern blot. Hybridization and detection of the telomeric sequences were performed as previously described [28 (link)]. DNA samples from K-562 cell line were used as internal control for telomere shortening, and cord blood cells as a control for no telomere reduction. In addition, peripheral blood mononuclear cells from 30 healthy individuals (17 males and 13 females; mean age 60.31 years, range: 31–86 years) with no personal or family history of cancer, matched by sex and age were also evaluated. All of them provided their informed consent.

Amplification procedure was performed by the use of 2 μl of the extracted DNA added to total mixture of 20 μl of amplification mixture supplied by Qiagen. The used primers were summarized in Table 1. The programmed cycles for amplifications were described previously by Villa et al., 1995 and Villa et al., 1999 [7 (link), 8 (link)].
Restriction Fragment length Polymorphism for Detection of Mutations in gyrA and parC.
HinfI (Promega) enzyme was used for the digestion of gyrA and parC in the amplified products of A. baumannii. The amplified products were purified by the use of QIA quick PCR purification kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. The eluant was incubated at 37°C for 2.5 hours with 10 U of HinfI. The digests were then separated by electrophoresis in 1.5% agarose gel stained with ethidium bromide and photographed with UV illumination. The presence of 291 bp indicated the presence of mutation in gyrA and the presence of 52 bp indicated the presence of mutation in parC.

An aliquot of extracted DNA was used to identify Anisakis genotypes using the mitochondrial cytochrome oxidase 2 (cox2) (~ 600 bp) and internal transcribed spacer (ITS) locus (~ 1000 bp, spanning through ITS1, 5.8S rRNA gene and ITS2). The latter amplicons were afterwards digested by restriction endonuclease HinfI (Promega, Madison, WI, USA) for restriction fragment length polymorphism analysis (RFLP-PCR) [27 (link)]. This step was necessary to exclude species other than A. pegreffii, potentially introduced into the striped dolphins while migrating from the Mediterranean.

The analysis of the ITS1-5.8S rDNA-ITS2 region was performed according to Csutak et al., [24 (link)]. The first step involved PCR amplification using specific primers ITS1 (5′-TCCGTAGGTGAACCTGCGG) and ITS4 (5′-TCCTCCGCTTATTGATATGC) (200 pM, TIB MOLBIOL). The amplicons were digested using four restriction endonucleases: CfoI (5′-GCG/C-3′), HinfI (5′-G/ANTC-3′), HaeIII (5′-GG/CC-3′), and MspI (5′-C/CGG-3′) (10 U/µL, Promega, Madison, WI, USA). The restriction fragments were observed by gel electrophoresis using 1.7% agarose and TBE 0.5X. The sizes of the amplicons and restriction fragments were determined using PyElph software [25 (link)]. Since for the isolates M1 and M5, the Biolog Microbial ID System and the MALDI-TOF MS analysis did not provide results, in order to confirm the data obtained using the conventional identification tests and the PCR-RFLP technique, the ITS1-5.8S rDNA-ITS2 amplicons were also sequenced. Sanger sequencing was performed by the CeMIA-Cellular and Molecular Immunological Applications Company (Larissa, Greece). The construction of contigs was done using the DNA Dragon 1.4.1 program [26 ]. Fasta sequences were uploaded on the GenBank database (Accession numbers: OP745444; OP745445). For the taxonomic identification of the isolates, the sequences obtained were compared with GenBank, using the BLAST algorithm [27 ].