Chromosome Identification via BAC-FISH

Preparation of slides and hybridization using bacterial artificial chromosome—fluorescent in situ hybridization (BAC-FISH) was carried as described in [53 (link)–55 (link)]. A B. distachyon BAC clone, ABR1-63-E6, that hybridizes all chromosomes of B. distachyon, but not those of B. stacei was labelled by random priming with biotin-14-dUTP (Invitrogen, Life Technologies) [56 (link)]. A ribosomal DNA probe, pTa 71,[57 (link)] which contains a 9-kb EcoRI fragment of rDNA repeat unit (18S-5.8S-26S genes and spacers) isolated from Triticum aestivum was labelled with Alexa-488 dUTP by random priming. Biotinylated probe was immunodetected using Texas Red avidin DCS (Vector Laboratories) and the signal was amplified with biotinylated anti-avidin D (Vector Laboratories). The chromosomes were mounted and counterstained in Vectashield (Vector Laboratories) containing 2.5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, Ariz) on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analysed using MetaVue^TM (Universal Imaging Corporation,Downington, PA).

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Dinh Thi V.H., Coriton O., Le Clainche I., Arnaud D., Gordon S.P., Linc G., Catalan P., Hasterok R., Vogel J.P., Jahier J, & Chalhoub B. (2016). Recreating Stable Brachypodium hybridum Allotetraploids by Uniting the Divergent Genomes of B. distachyon and B. stacei. PLoS ONE, 11(12), e0167171.

Publication 2016

biotin-14-dUTP Texas Red avidin DCS biotinylated anti-avidin D Vectashield Axioplan 2 microscope MetaVueTM

Avidin Bacterial artificial chromosome Biotin Chromosomes Chromosomes b Clone Dna probe Dutp Ecori Fluorescence Fluorescent in situ hybridization Genes Hybridization Microscope Rdna Ribosomal Triticum aestivum Vector

Corresponding Organization : Université Paris-Saclay

Top 5 similar protocols

Variable analysis

independent variables

Labelling of B. distachyon BAC clone ABR1-63-E6 with biotin-14-dUTP by random priming
Labelling of ribosomal DNA probe pTa 71 with Alexa-488 dUTP by random priming

dependent variables

Hybridization pattern of B. distachyon BAC clone ABR1-63-E6 on chromosomes
Hybridization pattern of ribosomal DNA probe pTa 71 on chromosomes

control variables

Preparation of slides and hybridization using bacterial artificial chromosome—fluorescent in situ hybridization (BAC-FISH) procedure as described in references [53–55]
Immunodetection of biotinylated probe using Texas Red avidin DCS and amplification with biotinylated anti-avidin D
Mounting and counterstaining of chromosomes in Vectashield containing DAPI

positive controls

B. distachyon BAC clone ABR1-63-E6 that hybridizes all chromosomes of B. distachyon, but not those of B. stacei

negative controls

Not explicitly mentioned

Annotations

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