Chromosome suspensions were sorted using an adaptation of a previously reported protocol60 (link) and a dual-laser cell sorter (MoFlo, Beckman Coulter), as performed at the Cambridge Resource Centre for Comparative Genomics (Cambridge, UK). Chromosome-specific painting probes were made by degenerate oligonucleotide primer PCR (DOP-PCR) amplification of flow-sorted chromosomes67 (link),68 (link). DOP-PCR amplified chromosome-specific DNAs were labeled during the secondary PCR by incorporating biotin-16-dUTP (Jena Bioscience) or digoxigenin-11-dUTP (Jena Bioscience). The PRO and PGO painting probes were generated as previously described69 (link).
Digoxigenin 11 dutp
Manufactured by Jena Biosciences
Sourced in United States
Digoxigenin-11-dUTP is a modified nucleotide commonly used in molecular biology applications. It serves as a labeling agent for the detection and localization of nucleic acid sequences through techniques such as in situ hybridization and Southern blotting.
Automatically generated - may contain errors
Lab products found in correlation
Biotin 16 dutp, by Jena Biosciences (4 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (1 mentions)
Aminoallyl dutp atto 647n, by Jena Biosciences (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
Geneart gene synthesis service, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
La taq dna polymerase, by Takara Bio (1 mentions)
Genomeplex wga reamplification kit, by Merck Group (1 mentions)
Genomeplex single cell whole genome amplification kit, by Merck Group (1 mentions)
Streptavidin alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti digoxigenin rhodamine, by Roche (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
7 protocols using digoxigenin 11 dutp
1
Chromosome Sorting and Painting Probe Generation
2
Chicken BAC-Clone Library FISH Probes
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Probes were prepared from the chicken BAC-clone library CHORI-261 (https://bacpacresources.org/chicken261.htm ) (Additional file 1 : Table S2). DNA was extracted from E.coli night cultures by standard alkaline-lysis protocol according to BAC manufacturer instructions. BAC DNA was labeled by nick-translation [58 ] at 16 °C for 2 h, using DNA polymerase I/DNAse I enzyme mix (ThermoFisher Scientific) and either biotin-11-dUTP (Lumiprobe), digoxigenin-11-dUTP (Jena Bioscience) or aminoallyl-dUTP-ATTO-647N (Jena Bioscience). Labeled probes were precipitated and dissolved at 20–30 ng/μl in hybridization mixture, containing 50% formamide, 10% dextran sulfate, 2 × SSC and 50 × excess of salmon sperm DNA (ThermoFisher Scientific). Since some BAC-clone based probes from the GGA1 gave unspecific hybridization signal on other chromosomes in metaphase plates, in case of these BAC clone combinations, 20 × excess of chicken Cot5 DNA prepared by S1-nuclease digestion was added to the hybridization mixture [59 ]. The probes were preannealed at 37 °C for 1 h after denaturation before mounting on slides.
3
Fluorescence In Situ Hybridization of Meiocytes
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Fluorescence in situ hybridization was carried out on prophase 1 meiocytes prepared from immature flower buds that were fixed in ethanol/acetic acid (3:1), following the protocol by Ross et al.38 (link). The following BAC clones were used: F9H3, T19B17, T26N6, F4H6, T4B21, T1J1 (IGF and TAMU library)39 (link),40 (link). The BAC DNA clones were labeled with either digoxigenin-11-dUTP or biotin-16-dUTP (Jena Bioscience GmbH) by nick translation following the manufacturer’s protocol (Sigma-Aldrich). In situ hybridization was carried out according to Lysak et al., 2006 with separate denaturation of probes and chromosomes41 . Microscopy slides were examined with a Zeiss AxioScope A1 fluorescence microscope using small band pass filters for DAPI, FITC, and Cy3. Images were captured with a Nikon color DS-Ri2 camera using Nikon NIS-elements 4.60 software. Microscope images were further processed with Adobe Photoshop software.
4
Functionalized DNA Handles for Assays
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Plasmid or λ phage DNA was used as template for PCR (Supplementary Table S1 ). Functionalized handles were amplified with a non-proofreading Taq DNA polymerase (New England Biolabs (NEB), Ipswich, MA USA.) and either biotin-16-dUTP or digoxigenin-11-dUTP (Jena Biosciences GmbH, Jena, Germany) was added to the PCR reactions to a final concentration of 40 μM, in addition to the 200 μM of non-labeled dNTPs. Other DNA fragments were obtained by PCR with the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), except for the fragment containing the stem-loop (S-SL), which was obtained with the LA Taq DNA polymerase (Takara Bio Europe) using the GC buffer I. PCR products were purified using either the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), the Wizard® SV Gel and PCR Clean-Up System (Promega GmbH, Mannheim, Germany) or the Monarch™ PCR & DNA Cleanup Kit (NEB). All primers were obtained from biomers.net. Unless otherwise stated, plasmids were obtained from the GeneArt Gene Synthesis Service (Thermo Fisher Scientific). Template DNA and primer design were performed with assistance of the molecular biology module of the Benchling platform.
5
Cross-Species Chromosome Painting in Xenopus
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We used whole chromosome painting probes generated by laser microdissection of X. tropicalis chromosomes from previous study by Knytl et al. (2023 ). Whole chromosome painting probes from X. tropicalis chromosomes 1 and 8 were newly labeled with digoxigenin-11-dUTP (Krylov et al. 2010 ) and biotin-16-dUTP (both Jena Bioscience) (Knytl et al. 2023 ), respectively. De-stained slides (after FISH with the U1 and U2 probes) of X. tropicalis, X. calcaratus, and X. laevis were used for cross-species painting FISH (Zoo-FISH) with a digoxigenin-labeled probe according to the protocols described in Krylov et al. (2010 ) and modified in Knytl et al. (2017 ). Subsequently, slides were de-stained again and used for Zoo-FISH with biotin-labeled probe. Detection of signal was carried as detailed for double-color painting in Knytl et al. (2023 ). The X. tropicalis FISH experiments were performed in the reverse order from the other species (i.e., painting FISH first followed by de-staining and snDNA FISH).
6
Whole Genome Painting for Xenopus Chromosomes
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Xenopus tropicalis genomic DNA (gDNA) was used as a probe for genomic in situ hybridization (GISH) experiments. Whole genome painting (WGP) probes were prepared using the GenomePlex Single Cell Whole Genome Amplification Kit (WGA4), Sigma-Aldrich, according to the manufacturer's whole genome amplification protocol with extracted gDNA. GenomePlex WGA Reamplification Kit (WGA3), Sigma-Aldrich, and labeling with Digoxigenin-11-dUTP (Jena Bioscience) was carried out as described in Krylov et al. (2010) (link). A combination of salmon sperm (Knytl, Kalous, Symonová, Rylková and Ráb, 2013b ) and autoclaved X. tropicalis gDNA (Bi and Bogart, 2006) (link) was used as a competitor DNA. Control GISH was performed on X. tropicalis chromosomes as detailed in painting FISH in Krylov et al. (2010) (link), and cross-species GISH was carried out on X. calcaratus chromosomes as detailed in Zoo-FISH in Krylov et al. (2010) (link) with minor changes described in Knytl et al. (2017) (link).
7
Multiprobe FISH Analysis of Vertebrate Karyotypes
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The FISH procedures were performed following Pinkel et al. (1986) protocol, with stringency ~77% (2.5 ng/μL probe, 50% formamide, 2x SSC, 10% dextran sulfate, at 37 °C for 16 h). It was used the following probes: 18S rDNA (Hatanaka, Galetti Junior, 2004), 5S rDNA (1200 bp DNA fragment amplified by PCR) and the general telomeric sequence of vertebrates (TTAGGG)n (Ijdo et al., 1991) . The probes 5S rDNA and (TTAGGG)n were labeled by PCR using digoxigenin 11-dUTP (Jena Bioscience); 18S rDNA probe was labeled with biotin through the nick translation technique ("Biotin16 NT Labeling Kit" -Jena Bioscience). For signal detection, the antibodies Streptavidin Alexa Fluor 488 (Molecular Probes) and antidigoxigenin-rhodamine (Roche Applied Science) were applied. Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI 0.2 μgmL -1 ) in mounting medium Vectashield (Vector) and analyzed under an epifluorescence microscope Olympus BX51, coupled to the Olympus DP-72 camera with the DP2-BSW software. The best images were photographed, and karyotypes edited using Adobe Photoshop CS6.
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