Fluorescence In Situ Hybridization for Glycine max Metaphase Chromosomes

Seth D. Findley1, James A. Birchler2, Gary Stacey1

1 University of Missouri, Division of Plant Sciences, Columbia, Missouri, 2 University of Missouri, Division of Biological Sciences, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20045
Online Posting Date:  March, 2017
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Abstract

This article presents protocols for fluorescence in situ hybridization (FISH) in the cultivated soybean, Glycine max. The protocols represent soybean‐optimized versions developed for maize. We describe the use of two different probes types: genomic‐repeat‐based fluorescently‐tagged oligonucleotides and bacterial artificial chromosomes (BACs). The two probe types can be used either individually or together, depending on the experimental questions. The article also includes starting points for executing FISH in additional legume species. © 2017 by John Wiley & Sons, Inc.

Keywords: bacterial artificial chromosomes (BACs); chromosome structure; fluorescence in situ hybridization (FISH); genomic in situ hybridization (GISH); Glycine max; karyotype; metaphase chromosomes; oligonucleotide probe; polyploidy; rolling‐circle amplification (RCA)

     
 
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Table of Contents

  • Introduction
  • Strategic Planning: Design of Single‐Stranded Fluorescent Oligonucleotide Probes
  • Basic Protocol 1: Fluorescence In Situ Hybridization
  • Support Protocol 1: Preparation and Use of Single‐Stranded Fluorescent Oligonucleotide Probes
  • Support Protocol 2: Preparation of FISH Probes from Rolling‐Circle‐Amplified BAC DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Fluorescence In Situ Hybridization

  Materials
  • Glycine max root‐tip metaphase chromosome slides (Findley et al., )
  • Sterile, high‐quality H 2O, such as double‐distilled (deionized distilled H 2O) or reverse‐osmosis (roH 2O) purified
  • 20× SSC (see recipe)
  • 10× TE buffer (see recipe)
  • 10 mg/ml sonicated salmon sperm DNA solution (e.g., Agilent, cat. no. 201190)
  • 5× single‐stranded oligonucleotide master cocktail solution ( protocol 2)
  • dsDNA probes ( protocol 3)
  • Aqueous mounting medium with DAPI (e.g., Vectashield with DAPI, Vector Laboratories)
  • Diamond‐tip pencil (e.g., Fisher, cat. no. 22‐268912)
  • Electric skillet with glass lid (e.g., Presto Model 06626 11‐in. electric skillet)
  • Fine‐tip forceps (e.g., Electron Microscopy Sciences 0508‐5XL‐PO) for handling plastic slides
  • Ice bucket
  • Thick aluminum plate that fits in the ice bucket, 12‐in. square (e.g., 0.16‐in. bare aluminum sheet, 2024 T3, http://www.onlinemetals.com)
  • Non‐aluminum metal cake pan, 8 in. × 8 in. × 2 in. (e.g., Wilton brand Perfect Results 8‐in. square cake pan)
  • Square plastic disposable coverslips (22 mm; e.g., Fisher, cat. no. 12‐547)
  • Disposable plastic food container (e.g., Glad brand, sandwich size) that fits in the metal cake pan, above
  • 0.2‐ml thin‐wall PCR tubes
  • Thermal cycler
  • Hybridization tray and rack for slides (e.g., Boekel, cat. no. C2403973 and C2403754)
  • Hybridization oven (e.g., UVP HB‐1000, or other reliable 55°C oven)
  • Large rubber bands
  • Glass slide washing chambers (e.g., Wheaton 900303 Soda Lime Glass Staining Dish)
  • Microwave oven
  • 250‐ or 300‐ml beaker
  • Square glass coverslips
  • Light microscope with digital camera

Support Protocol 1: Preparation and Use of Single‐Stranded Fluorescent Oligonucleotide Probes

  Materials
  • Lyophilized 5′‐end‐fluor‐labeled oligonucleotides (e.g., Integrated DNA Technologies; IDT)
  • Sterile, double‐distilled or reverse‐osmosis H 2O (distilled deionized H 2O)
  • 10× TE buffer (see recipe)
  • 20× SSC (see recipe)
  • Black or amber 0.5‐ml microcentrifuge tubes (e.g., LiteSafe Micro‐Tubes from Research Products International)
  • Ice bucket

Support Protocol 2: Preparation of FISH Probes from Rolling‐Circle‐Amplified BAC DNA

  Materials
  • Bacterial artificial chromosome (BAC) miniprep DNA(s) growing on selective medium
  • Sterile, double‐distilled or reverse‐osmosis water (ddH 2O)
  • LB medium (see recipe)
  • Chloramphenicol
  • Commercial DNA purification kit (e.g., Promega Wizard Plus SV Miniprep kit)
  • 2× RCA annealing buffer (see recipe)
  • Solution of 1 mM thiophosphate‐modified random hexamer primers (“TRP solution”; Integrated DNA Technologies)
  • 40 mM dithiothreitol (freshly diluted from 1 M stock)
  • 4 mM dNTP working solution (i.e., single solution, 4 mM concentration of each dNTP)
  • 10× Phi29 DNA polymerase buffer (New England Biolabs)
  • Phi29 DNA Polymerase (New England Biolabs, cat. no. M0269S, 10,000 U/ml)
  • 10× nick‐translation buffer (see recipe)
  • 2 mM [‐C] dNTP mix (or 2 mM [‐T] dNTP mix, if fluorescein‐12‐dUTP is used) (see recipe)
  • Texas Red‐5‐dCTP (e.g., Perkin‐Elmer, cat. no. NEL426001EA); alternatively, fluorescein‐12‐dUTP (e.g., Perkin‐Elmer, cat. no. NEL413001EA)
  • DNA Polymerase I (New England Biolabs, cat. no. M0209L)
  • DNase I (New England Biolabs, cat. no. M0303S or Sigma‐Aldrich, cat. no. 4716728001)
  • 10× TE buffer (see recipe)
  • 20× SSC (see recipe)
  • Thin‐walled 0.2‐ml PCR tubes
  • Ice bucket
  • Nanodrop microspectrophotometer
  • Thermal cycler
  • Black or amber 0.5‐ml microcentrifuge tubes (e.g., LiteSafe Micro‐Tubes from Research Products International)
  • Vacuum centrifuge (e.g., Speedvac)
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Figures

Videos

Literature Cited

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