In Situ Hybridization in Rice (Oryza sativa)

Kai Wang1, Weichang Yu2

1 Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fujian, China, 2 Shenzhen Research Institute of the Chinese University of Hong Kong, Shenzhen, China
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20007
Online Posting Date:  May, 2016
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Abstract

Fluorescence in situ hybridization (FISH) is widely used in cytogenetics to determine the localization of DNA sequences on target chromosomes, to provide visible information regarding the physical position of DNA sequences, to determine the abundance and distribution of repetitive sequences that comprise a large proportion of genomes, and to determine the relative chromosome positions of multiple sequences in physical mapping. By mapping on extended chromatin fibers, fiber‐FISH can be used to determine the structure and organization of genes or DNA sequences with a high resolution (to a few kilobases). The protocols described here will provide procedures of FISH on metaphase chromosomes and extended chromatin fibers of rice (Oryza sativa). © 2016 by John Wiley & Sons, Inc.

Keywords: fluorescence in situ hybridization (FISH); metaphase chromosome; chromatin fiber; fiber‐FISH; rice

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Direct Fluorescence In Situ Hybridization in Rice Metaphase Chromosomes using Fluorescent Probes
  • Alternate Protocol 1: Indirect Fluorescence In Situ Hybridization in Rice Metaphase Chromosomes Using Hapten‐Labeled Probes
  • Alternate Protocol 2: Fiber‐FISH: Hybridization of FISH Probes to Extended Chromatin Fibers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Direct Fluorescence In Situ Hybridization in Rice Metaphase Chromosomes using Fluorescent Probes

  Materials
  • Slides with mitotic chromosomes prepared from rice root tips (Wang and Yu, )
  • Plasmid containing rice centromere repeat CentO (available from W. Yu)
  • Miniprep kit (Qiagen)
  • 10× nick translation buffer I (see recipe)
  • Chroma Tide Alexa Fluor 594‐5‐dUTP (Life Technologies)
  • 2 mM dNTP mix: 2 mM each dATP, dGTP, and dCTP
  • 10 U/μl DNA polymerase I (Life Technologies)
  • 100 U/ml DNase I (see recipe)
  • 50× TAE buffer (see recipe)
  • 10 mg/ml salmon sperm DNA (ssDNA; see recipe)
  • 3 M sodium acetate, pH 5.2 (see recipe)
  • Absolute ethanol
  • 2× SSC (see recipe)
  • Vectashield mounting medium with 4′,6‐diamidino‐2‐phenylindole (DAPI; Vector Laboratories)
  • UV crosslinker
  • Glass cutter
  • 0.5‐ and 1.5‐ml black microcentrifuge tubes
  • Thermocycler
  • 95°C heating block
  • Agarose gel electrophoresis apparatus with 2% (w/v) gel
  • Airtight plastic container
  • Incubator at 55°C
  • Aluminum foil
  • Square electric cooker
  • 24 × 32–mm coverslips
  • Blotting paper
  • Metal box with lid (e.g., stainless steel food container)
  • Coplin jars
  • Fluorescence microscope equipped with appropriate filter sets and a CCD camera
  • Image processing software (e.g., Adobe Photoshop)

Alternate Protocol 1: Indirect Fluorescence In Situ Hybridization in Rice Metaphase Chromosomes Using Hapten‐Labeled Probes

  Additional Materials (also see protocol 1Basic Protocol)
  • Separate plasmids encoding rice 45S and 5S rDNA (available from W. Yu)
  • 10× nick translation buffer II (see recipe)
  • Biotin‐ and digoxigenin‐labeled dUTP/dTTP mixes (see recipe)
  • 0.5 mM dNTP mix: 0.5 mM each dATP, dGTP, dCTP
  • 3.3 ng/μl DNase I (see recipe)
  • 0.5 M EDTA, pH 8.0
  • Deionized formamide (Sigma)
  • 50% (w/v) dextran sulfate (see recipe)
  • C 0t‐1 DNA (optional; see Strategic Planning)
  • 70%, 90%, and 100% (v/v) ethanol, –20°C
  • 1× PBS (see recipe)
  • Alexa Fluor 488–streptavidin (1 mg/ml, Life Technologies), reconstituted in 1× PBS, aliquot and store at −20°C in the dark
  • Rhodamine‐anti‐digoxigenin (200 μg/ml, Roche), reconstituted in ddH 2O, aliquot and store at −20°C in the dark
  • TNB buffer (see recipe)
  • 0.5‐ml PCR tubes
  • Mini‐microcentrifuge
  • G50 columns (Roche)
  • 85°C heating block
  • 70°C hot plate
  • Rubber cement
  • 37°C incubator
  • Forceps

Alternate Protocol 2: Fiber‐FISH: Hybridization of FISH Probes to Extended Chromatin Fibers

  Additional Materials (also see protocol 2)
  • Young rice leaves
  • Liquid nitrogen
  • Nucleus isolation buffer (see recipe)
  • 2‐Mercaptoethanol (Sigma)
  • Triton X‐100 (Sigma)
  • Nucleus stock buffer: 50% (v/v) glycerol and 50% nucleus isolation buffer
  • Carnoy's fixative (see recipe)
  • STE lysis buffer (see recipe)
  • Plasmids with desired probe sequences (e.g., 45S rDNA, CentO, and centromere retrotransposon of rice [CRR]; all available from W. Yu)
  • 1× TNT (see recipe)
  • Biotinylated anti‐streptavidin antibody (500 μg/ml, Vector Labs), reconstituted in 1× PBS, aliquot and store at −20°C
  • Mouse anti‐digoxigenin antibody (100 μg/ml, Roche), reconstituted in 1× PBS, aliquot and store at −20°C
  • Alexa Fluor 568–rabbit anti‐mouse antibody (Life Technologies), aliquot and store at −20°C in the dark
  • Vectashield mounting medium without DAPI (Vectashield)
  • Mortar and pestle
  • 50‐ml centrifuge tubes
  • Horizontal rotary shaker
  • Mira cloth (Calbiochem)
  • Nylon membranes (30‐ and 50‐μm pore sizes, Sefar Nylon Mesh Lab Pak)
  • 1.5‐ml microcentrifuge tubes
  • Poly‐L‐lysine‐coated slides (Sigma)
  • 18 × 18–mm coverslips
  • 65°C hot plate
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Figures

Videos

Literature Cited

Literature Cited
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