Cy3 dutp

A human digoxigenin-labeled telomere probe was purchased from Appligene Oncor (Lifescreen, Watford, UK). Random prime labeling of the probe DNA from PCR products was performed with

fluorescein-12-dUTP

(F-dUTP) or

cyanine-3-dUTP

(Cy3-dUTP) in accordance with the kit protocol (Invitrogen, Tokyo, Japan). FISH was conducted as previously reported (Takaoka et al. 2012 (link)), with slight modifications. In brief, the metaphase preparations were denatured in 70% formamide/2× saline-sodium citrate at 73 °C for 3 min. Then 1 μl of probe was mixed with 10 μl of hybridization solution (H7782, Sigma, Japan) and denatured at 80 °C for 10 min. HybridizaPageBreaktion of the probes was performed at 37 °C in the CO₂ incubator for 12–15 h, followed by post-hybridization washes, DAPI

(4’,6-diamidino-2-phenylindole)

counterstaining and visualization of the probes under a fluorescence microscope (Olympus BX50).

Target genes (18S rRNA, U2 snRNA-5S, histone H3, and uncharacterized gene FP-9X) were amplified by PCR using the Emerald PCR master mix (Takara, Japan). The primer sets used are shown in Table 1. PCR was performed in a thermal cycler (WK-0518, Wako, Osaka, Japan) under the following conditions: initial denaturation for 2 min at 98 °C, followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min. PCR products were ligated into the pGEM-T Easy Vector (Promega, Madison, USA) and 30 ng of the ligation products were used to transform competent cells (JM109, pGMT-T Easy-Vector Systems, Promega). Transformed cells were plated onto Luria broth (LB) plates containing 100 μg/ml ampicillin, 40 μg/ml 5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside (X-Gal) and 0.05 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG). Isolated colonies were screened by FISH. Probes using FISH screening were prepared by random prime labeling with digoxigenin-12-dUTP (DIG-dUTP) or cyanine-3-dUTP (Cy3-dUTP) in accordance with the kit protocol (Invitrogen, Tokyo, Japan). Then, FISH-positive clones were later transferred into 15 ml test tubes containing 1.5 ml of LB/ampicillin medium and grown at 37 °C overnight. Plasmids from the resulting clones were extracted according to the manufacturer’s protocol using a Mini Plus Plasmid DNA Extraction System (Viogene, NACALAI TESQUE, INC., Kyoto, Japan).