Multi-color FISH Analysis of Canine BAC Clones

A total of 851 BAC clones from the RH map were also localized by multi-color FISH analysis. DNA from each clone was prepared from 2.5 ml cultures using a BAC RealPrep (Qiagen, Valencia, CA) protocol. Two hundred nanograms from each sample were labeled using nick translation to incorporate one of five fluorochromes, Spectrum Red/Orange/Green dUTP (Vysis, Downers Grove, IL), diethylaminomethylcoumarin (DEAC)-5-dUTP (NEN/Perkin Elmer Life Sciences, Boston, MA), or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ). Typically, 25 ng of each of five differentially labeled probes were pooled and precipitated in the presence of 15 μg of sonicated genomic dog DNA as competitor. Chromosome preparation, probe hybridization and post hybridization washes were performed as described previously [35 (link),36 (link)]. Chromosomes were counterstained in 80 ng/ml 4', 6-diamidino-2-phenylindole (DAPI) and mounted in anti-fade solution (Vectashield, Vector Laboratories, Burlingame, CA). Images were acquired and processed using a multi-color FISH workstation comprising a fluorescence microscope (Axioplan 2ie, Zeiss) equipped with narrow pass filter sets and a cooled CCD camera (CoolSnapHQ, Photometrics, Tuscon, AZ) both driven by dedicated software (SmartCapture 2.3.1 Digital Scientific, Cambridge, U.K.). The digital image of each DAPI stained metaphase spread was processed using a high-pass spatial filter to reveal enhanced DAPI bands. Clones were assigned to a chromosome region according to the DAPI banded nomenclature of Breen et al. [35 (link),36 (link)]. Refinement of probe order along the length of each chromosome was made by subsequent rehybridization to elongated canine chromosome preparations and/or by reference to interphase FISH analysis. Additional information may be found at .

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Breen M., Hitte C., Lorentzen T.D., Thomas R., Cadieu E., Sabacan L., Scott A., Evanno G., Parker H.G., Kirkness E.F., Hudson R., Guyon R., Mahairas G.G., Gelfenbeyn B., Fraser C.M., André C., Galibert F, & Ostrander E.A. (2004). An integrated 4249 marker FISH/RH map of the canine genome. BMC Genomics, 5, 65.

Publication 2004

Canine Chromosome Clones Cy5 dutp Dutp Fish Fluorescence microscope Fluorochromes Genomic Hybridization Interphase Metaphase Spectrum orange Vector

Corresponding Organization :

Other organizations : North Carolina State University, Centre National de la Recherche Scientifique, Cape Town HVTN Immunology Laboratory / Hutchinson Centre Research Institute of South Africa, Animal Health Trust

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Protocol cited in 13 other protocols

Variable analysis

independent variables

The protocol used to prepare DNA from each BAC clone (BAC RealPrep protocol from Qiagen)
The amount of DNA used for labeling (200 ng from each sample)
The labeling method used (nick translation to incorporate one of five fluorochromes: Spectrum Red/Orange/Green dUTP, DEAC-5-dUTP, or Cy5-dUTP)
The amount of differentially labeled probes pooled and precipitated (25 ng of each of five probes)
The addition of 15 μg of sonicated genomic dog DNA as competitor

dependent variables

The localization of the 851 BAC clones from the RH map by multi-color FISH analysis
The chromosomal region assignment of the clones according to the DAPI banded nomenclature

control variables

Chromosome preparation, probe hybridization, and post-hybridization washes were performed as described previously in the referenced publications [35, 36]
Chromosomes were counterstained in 80 ng/ml 4', 6-diamidino-2-phenylindole (DAPI) and mounted in anti-fade solution (Vectashield)
The digital image of each DAPI-stained metaphase spread was processed using a high-pass spatial filter to reveal enhanced DAPI bands

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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