Avidin fitc

The protocol for selection and coordinates of BAC-clones was reported in previous research^{20 (link)}. The list of BAC-clones from CHORI-240 library is shown in table (Suppl. 3). BAC clones’ DNA was isolated using the Plasmid DNA Isolation Kit (BioSilica, Novosibirsk, Russia) and amplified with GenomePlex Whole Genome Amplification kit (Sigma-Aldrich Co., St. Louis, MO, USA). Labeling of BAC clone DNA was performed using GenomePlex WGA Reamplification Kit (Sigma-Aldrich Co., St. Louis, MO, USA) by incorporating biotin-16-dUTP or digoxigenin-dUTP (Roche, Basel, Switzerland).
Dual-color FISH experiments were conducted as described by Yang and Graphodatsky⁴⁶ . Trypsin-treated chromosomes were immobilized in 0.5% formaldehyde in PBS followed by formamide denaturing and overnight probe hybridization at 40◦C. Digoxigenin-labeled probes were detected using anti-digoxigenin-CyTM3 (Jackson Immunoresearch), whereas biotin-labeled probes were identified with avidin-FITC (Vector Laboratories) and anti-avidin FITC (Vector Laboratories). Images were captured and processed using VideoTesT 2.0 Image Analysis System and a Baumer Optronics CCD Camera mounted on an Olympus BX53 microscope (Olympus).

Mitotic metaphases were obtained from bone marrow cells [53 (link)]. Chromosome slides were prepared according to the standard air-drying technique. The C-banding technique was performed as described by Sumner [54 (link)]. To obtain G-banded chromosomes, we used a simple two-step method (modified method from Seabright [55 (link)]). For the localization of telomeric sequences, we used commercially available telomeric (TTAGGG)n probes labeled with biotin-16-dUTP (Roche Biomolecular, Indianapolis, IN, USA), detected with avidin-FITC (Vector Laboratories, Burlingame, CA, USA). The hybridization was carried out at 37 °C overnight. The chromosomes were counterstained with DAPI.

Dual-color FISH experiments on G-banded metaphase chromosomes were conducted as described by Yang and Graphodatsky [32 ]. Tripsin-treated chromosomes were immobilized in 0.5% formaldehyde in PBS followed by formamid denaturing and overnight probe hybridization at 40 °C. Digoxigenin-labeled probes were detected using anti-digoxigenin-CyTM3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, whereas biotin-labeled probes were identified with avidin-FITC (Vector Laboratories) and anti-avidin FITC (Vector Laboratories, Inc., Burlingame, CA, USA). Images were captured and processed using VideoTesT 2.0 Image Analysis System (Zenit, St. Petersburg, Russia) and a Baumer Optronics CCD camera mounted on a BX53 microscope (Olympus, Shinjuku, Japan).

The probes for CGH were prepared by nick translation (Abbott Laboratories, Lake Bluff, IL, USA), according to the manufacturer’s protocol. The probe specific for male genome was labeled by dUTP-biotin (Roche, Basel, Switzerland), while the probe specific for female genome by dUTP-digoxigenin (Roche, Basel, Switzerland). Both probes were mixed in equal concentration and diluted in hybridization buffer (50% formamide in 2 × SSC; 10% dextran sulfate; 10% sodium dodecyl sulfate; and Denhard’s buffer, pH 7.0). The FISH protocol follows the protocol of the FISH with telomeric probe for the slide preparation. The slides were incubated for 48 h at 37 °C. The probe was washed in 50% formamide/2 × SSC three times for 5 min at 37 °C. In the next step, the slides were washed in 2 × SSC at room temperature and 4 × SSC/0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). After the washes, we incubated the slides in 4 × SSC/5% blocking reagent for 30 min at 37 °C. Subsequently, we incubated the slides with avidin-FITC (Vector Laboratories, Burlingame, CA, USA) and anti-digoxigenin-Rhodamin (Roche, Basel, Switzerland) for 30 min at 37 °C. The slides were washed in 4 × SSC/0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) and PBS solution and dehydrated in ethanol series. The slides were stained by Fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, USA).

BAC DNA labeling, hybridizations and signal detection were carried out according to standard protocols (Raudsepp and Chowdhary, 2008 (link)). The DNA of individual BACs was labeled with biotin or digoxigenin using DIG- or Biotin-Nick Translation Mix (Roche Diagnostics) and the manufacturer’s protocol. Because the known difficulties to unambiguously identify camelid chromosomes, we consulted Zoo-FISH data (Balmus et al., 2007 ) and the 230-marker cytogenetic map (Avila et al., 2014a (link)) to infer the most probable chromosome location for each candidate gene. Based on these predictions, BACs containing new genes were co-hybridized with a differently labeled reference gene from the cytogenetic map (Table 2). Biotin- and dig-labeled probes were detected with avidin-FITC (Vector Laboratories) and anti-dig-rhodamine (Roche Applied Science), respectively. Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and identified according to the nomenclature proposed by Balmus et al. (2007) and Avila et al. (2014b) (link). Images were captured and analyzed using a Zeiss Axioplan 2 fluorescence microscope, equipped with the Isis Version 5.2 (MetaSystems GmbH) software. At least 10 images were captured and analyzed for each experiment.