Orange dutp

Bovine whole-chromosome painting (WCP) probes were used for identification of chromosomes involved in the Robertsonian and Tandem fusions in animals analyzed in this study. Bovine whole chromosomes were isolated by flow sorting using MoFlo XDP Cell Sorter (Beckman Coulter, Brea, CA, USA) [29 (link)] or microdissected by PALM Microlaser system (Carl Zeiss MicroImaging GmbH, Munich, Germany) [30 (link)]. Once isolated, bovine chromosomes were used to produce WCP probes by DOP-PCR [31 (link)]. Probe labeling was performed during the secondary PCR with Green-dUTP or Orange-dUTP (Abbott Park, Chicago, IL, USA) [30 (link)].
For sperm-FISH, bovine BAC clones localized to the chromosomes involved in translocations were selected from the CHORI-240 cattle library (BACPAC Genomics, Emeryville, CA, USA). BAC DNA labeling was with digoxigenin-11-dUTP or biotin16-dUTP (Roche, Mannheim, Germany) was performed using BioPrime Array CGH Genomic Labeling Module (Invitrogen, Carlsbad, CA, USA). Detailed list of BACs used in the present study appears in Table S1.

Whole chromosome painting probes from cattle (BTA1-29) were used for cross-species hybridization to RSH, RCA and RME. The orientation of the syntenic blocks comprising RSH1, RCA1 and RME1 (the largest fusion chromosome in Raphicerus) was by region-specific paints BTA25qd, BTA14qd and BTA1qd. Analysis of the X chromosomes relied on arm- and region-specific painting probes from cattle and goat^{41 (link)} that localized to BTAXp, BTAXq, BTA Xq 3.6-qter and CHI Xq 4.1-qter respectively. Y chromosomes were examined using a painting probe originally prepared from Kirk´s dik-dik (Madoqua kirkii, MKI^{40 (link)}). Detection of Nucleolar Organizer Regions (NORs) was by FISH probes prepared from the antelope, Nanger dama^{41 (link)}.
In all instances painting probes were prepared by laser microdissection (PALM Microlaser system, Carl Zeiss MicroImaging GmbH, Munich, Germany) and DNA amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR: primer sequence CCGACTCGAGNNNNNNATGTGG)^{64 (link)}. Labelling during the secondary PCR^{64 (link)} was with Orange-d UTP or Green-dUTP (Abbott, IL, USA).

Whole chromosomes or chromosomal regions for the construction of painting probes were isolated by flow sorting using MoFlo XDP Cell Sorter (Beckman Coulter, USA) [20 ] or microdissected by PALM Microlaser system (Carl Zeiss MicroImaging GmbH, Munich, Germany). The chromosomal DNA was then amplified by DOP-PCR (degenerate oligonucleotide primed polymerase chain reaction) [38 (link)]. Probe labelling was performed during the secondary PCR with Green-dUTP or Orange-dUTP (Abbott, IL, USA) [39 (link)].

We used the PALM Microlaser system (Carl Zeiss MicroImaging GmbH, Munich, Germany) to collect 20 copies of the marker chromosome. DNA of the collected chromosomes was amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR), labeled during the secondary PCR with Orange-dUTP (Abbott, IL, USA) as described by Kubickova et al. [24 (link)] and checked by FISH. Amplification products derived from the marker were cloned into a pDrive vector (Qiagen, Hilden, Germany). The clones were screened by DOT-BLOT hybridization [25 (link)], fluorescently labeled by Orange-dUTP, and checked for specificity by FISH. Plasmid DNA of the selected clone was subsequently isolated and sequenced by Sanger sequencing. The clone comprised repetitive DNA but was not long enough to represent a basic repeat unit (BRU). Therefore, primers amplifying the 5′- and 3′- flanking regions were designed and inverse PCR was performed on the genomic DNA [20 ]. The amplification products representing the BRU obtained by PCR were cloned and the plasmid DNA was isolated, fluorescently labeled by Orange-dUTP, and used in the FISH analysis. The BRU clone was named EMAM1 clone, sequenced, and deposited in GenBank under accession number OP918028.