Fluorescence in situ hybridization (FISH) was performed to map repeated DNAs on the mitotic and meiotic chromosomes of H. obliquidens. Five DNA probes containing sequences of different classes of repeated DNA were used for chromosome hybridization. (i) 5S rDNA probe: complete repeat units of 5S rDNA of H. obliquidens were obtained by the polymerase chain reaction (PCR) with the primers 5SA (5'-TAC GCC CGA TCT CGT CCG ATC - 3') and 5SB (5' - CAG GCT GGT ATG GCC GTA AGC-3') designed from the rainbow trout 5S rRNA sequence [63 (link)] and successfully applied for the amplification of 5S rDNA of other cichlids [64 (link),65 (link)]. (ii) 18S rDNA probe: a segment of 1,400 base pairs (bp) of the 18S rRNA gene of H. obliquidens was obtained by PCR with the primers 18Sf 5'CCG CTT TGG TGA CTC TTG AT and18Sr 5'CCG AGG ACC TCA CTA AAC CA. The 18S primers were designed from the catfish Ictalurus punctatus (GenBank accession number AF021880 ) and have been successfully used to amplify 18S rRNA genes of different fish species [65 (link),66 (link)]. (iii) SATA satellite: repeated satellite DNA isolated and cloned from O. niloticus [29 (link)]; (iv) Telomeric DNA sequences: in vitro synthesized oligomers of telomeric repeats (GGGTTA)7/(TAACCC)7; (v) Clones BAC-C4E09 and BAC-C5E01: Bacterial artificial chromosomes containing several classes of repeated elements from the O. niloticus genome [29 (link)].
Probes were labeled by nick translation with biotin 14-dATP (Bionick labeling system-Invitrogen). After denaturation of chromosomal DNA in 70% formamide/2× SSC for 40 seconds at 70°C, hybridization mixtures containing 100 ng of denatured probe, 10 mg/ml dextran sulfate, 2× SSC and 50% formamide, in a final volume of 30 μl, were dropped on the slides and the hybridization was performed overnight at 37°C in a 2× SSC moist chamber. Post-hybridization washes were carried out at 45°C in 2× SSC/50% formamide for 15 min, followed by a second wash in 2× SSC for 15 min, and a final wash at room temperature in 4× SSC for 15 min. Detection of hybridized probes was carried out with 0.07% avidin FITC conjugate (Sigma) in C buffer (0.1 M NaHCO3 /0.15 M NaCl) for 1 h, followed by two rounds of signal amplification using 2.5% anti-avidin biotin conjugate (Sigma) in blocking buffer (1.26% NaHCO3, 0.018% sodium citrate, 0.0386% Triton X-100 an 1% non-fat dried milk) for 30 min. Each treatment with anti-avidin biotin conjugate was followed by a treatment with avidin-FITC. The treatments with avidin-FITC and anti-avidin-biotin were conducted in a 2× SSC moist chamber at 37°C. After each amplification step, the slides were washed three times for 5 min each in blocking buffer at 42°C. Chromosomes were counterstained with propidium iodide diluted in antifade (Vectashield Mounting Medium, Vector). Hybridized chromosomes were visualized using an Olympus BX 61 microscope, and images were captured with a digital camera Olympus DP71 with the software Image-Pro MC 6.0. Karyotypes and metaphases were arranged with Adobe Photoshop 7.0 software.
Probes were labeled by nick translation with biotin 14-dATP (Bionick labeling system-Invitrogen). After denaturation of chromosomal DNA in 70% formamide/2× SSC for 40 seconds at 70°C, hybridization mixtures containing 100 ng of denatured probe, 10 mg/ml dextran sulfate, 2× SSC and 50% formamide, in a final volume of 30 μl, were dropped on the slides and the hybridization was performed overnight at 37°C in a 2× SSC moist chamber. Post-hybridization washes were carried out at 45°C in 2× SSC/50% formamide for 15 min, followed by a second wash in 2× SSC for 15 min, and a final wash at room temperature in 4× SSC for 15 min. Detection of hybridized probes was carried out with 0.07% avidin FITC conjugate (Sigma) in C buffer (0.1 M NaHCO3 /0.15 M NaCl) for 1 h, followed by two rounds of signal amplification using 2.5% anti-avidin biotin conjugate (Sigma) in blocking buffer (1.26% NaHCO3, 0.018% sodium citrate, 0.0386% Triton X-100 an 1% non-fat dried milk) for 30 min. Each treatment with anti-avidin biotin conjugate was followed by a treatment with avidin-FITC. The treatments with avidin-FITC and anti-avidin-biotin were conducted in a 2× SSC moist chamber at 37°C. After each amplification step, the slides were washed three times for 5 min each in blocking buffer at 42°C. Chromosomes were counterstained with propidium iodide diluted in antifade (Vectashield Mounting Medium, Vector). Hybridized chromosomes were visualized using an Olympus BX 61 microscope, and images were captured with a digital camera Olympus DP71 with the software Image-Pro MC 6.0. Karyotypes and metaphases were arranged with Adobe Photoshop 7.0 software.
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