Visualizing Genomes with Oligopaint FISH Probes

Brian J. Beliveau1, Nicholas Apostolopoulos2, Chao‐ting Wu1

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Yale School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.23
DOI:  10.1002/0471142727.mb1423s105
Online Posting Date:  January, 2014
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Abstract

Oligopaint probes are fluorescently labeled, single‐stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. This unit details how Oligopaint probes can be synthesized using basic molecular biological techniques, and provides protocols for FISH, 3D‐FISH, and sample preparation. Curr. Protoc. Mol. Biol. 105:14.23.1‐14.23.20. © 2014 by John Wiley & Sons, Inc.

Keywords: Oligopaint; oligonucleotide; complex DNA library; FISH

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Single‐Stranded Oligopaint FISH Probes from a Complex DNA Library
  • Basic Protocol 2: Interphase FISH Using Oligopaint Probes
  • Alternate Protocol 1: Fast 3D‐FISH Using Oligopaint Probes
  • Alternate Protocol 2: Metaphase FISH with Oligopaint Probes
  • Support Protocol 1: Preparing Tissue Culture Cells for Interphase FISH
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of Single‐Stranded Oligopaint FISH Probes from a Complex DNA Library

  Materials
  • 10× Taq DNA polymerase buffer (KAPA Biosystems)
  • 10 mM dNTP mix
  • 200 µM fluorophore‐labeled “forward” primer (Integrated DNA Technologies)
  • 200 µM unlabeled “reverse” primer (Integrated DNA Technologies)
  • 20 pg/µl complex DNA library (MYcroarray, LC Sciences, CustomArray)
  • 5 U/µl Taq DNA polymerase (KAPA Biosystems)
  • Molecular biology‐grade water
  • 20 mg/ml molecular biology‐grade glycogen (Thermo Scientific)
  • 4 M ammonium acetate solution in distilled, deionized water (ddH 2O)
  • 100% ethanol
  • 70% (v/v) ethanol solution in ddH 2O
  • 10× Nb.BsrDI enzyme buffer (nicking enzyme buffer; New England Biolabs)
  • 10 U/µl Nb.BsrDI enzyme (New England Biolabs)
  • 1× Tris‐Borate‐EDTA (TBE) buffer (see recipe)
  • 15% TBE + 7 M Urea denaturing polyacrylamide gel (Bio‐Rad)
  • Low‐molecular‐weight DNA ladder
  • 2× TBE + urea gel loading buffer containing xylene cyanol FF and bromphenol blue (Bio‐Rad)
  • Ice
  • 10 mg/ml ethidium bromide solution (Calbiochem)
  • 0.4 M ammonium acetate solution in ddH 2O
  • 0.2‐ml thin‐walled strip tubes or thin‐walled 96‐well plate
  • Programmable thermal cycler
  • 50‐ml conical tubes
  • Benchtop vortex mixer
  • 2.0‐ml microcentrifuge tubes
  • −80°C or −20°C freezer
  • Refrigerated centrifuge
  • Vacuum trap or micropipets
  • Adjustable heat block
  • 37°C incubator
  • Microcentrifuge
  • 15‐ml conical tubes
  • Gel box and power supply
  • 1000‐µl micropipets
  • Ethidium bromide staining dish
  • Benchtop orbital shaker
  • UV box
  • Heated vortex mixer or shaking incubator
  • Spectrophotometer

Basic Protocol 2: Interphase FISH Using Oligopaint Probes

  Materials
  • 4× SSCT (see recipe)
  • Formamide (store at 4°C, away from light)
  • Fixed interphase cells adhered to a glass microscope slide (see Support Protocol)
  • 2× hybridization cocktail (see recipe)
  • 10 mg/ml RNase A
  • Oligopaint probe (see protocol 1)
  • 2× SSCT (see recipe)
  • 0.2× SSC (see recipe)
  • Anti‐fade mounting medium with DAPI
  • Nail polish
  • 100‐ml graduated cylinders
  • Plastic paraffin film
  • Glass Coplin slide staining jars
  • Adjustable temperature water baths
  • Anodized aluminum heat block
  • Forceps
  • 1.7‐ml microcentrifuge tubes
  • Benchtop vortex mixer
  • Benchtop microcentrifuge
  • Aluminum foil
  • Paper towels
  • 22 × 22–mm no. 1.5 coverslips
  • Pipets
  • Rubber cement
  • Hybridization chamber (see step 15)
  • Heated incubator
  • 22 × 30–mm no. 1.5 coverslips
  • Epifluorescent or confocal microscope

Alternate Protocol 1: Fast 3D‐FISH Using Oligopaint Probes

  Additional Materials (also see protocol 2)
  • 1× PBS (see recipe)
  • 1× PBST (see recipe)
  • 1× PBS + 0.5% (v/v) Triton‐X100
  • 0.1 N HCl (625 µl of 8 N HCl in 50 ml ddH 2O)

Alternate Protocol 2: Metaphase FISH with Oligopaint Probes

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • Sample slide containing spread mitotic chromosomes
  • 70% (v/v) ethanol in ddH 2O
  • 90% (v/v) ethanol in ddH 2O
  • 100% (v/v) ethanol

Support Protocol 1: Preparing Tissue Culture Cells for Interphase FISH

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • 0.01% (v/v) poly‐L‐lysine solution in ddH 2O (5 ml of 0.1% stock + 45 ml ddH 2O)
  • 1 × 105–1 × 106 cells/ml cell suspension
  • Complete growth medium
  • 1× PBS + 4% (v/v) paraformaldehyde (see recipe)
  • Lint‐free paper towels (e.g., Kimwipes)
  • 25 × 75 × 1–mm glass microscope slides
  • Plastic Coplin staining jar
  • Cell culture incubator
  • Micropipets
NOTE: All steps are performed at room temperature unless otherwise indicated.
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Figures

Videos

Literature Cited

  Bauman, J.G., Wiegant, J., Borst, P., and van Duijn, P. 1980. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of flurochromelabelled RNA. Exp. Cell. Res. 128:485‐490.
  Beliveau, B.J., Joyce, E.F., Apostolopoulos, N.A., Yilmaz, F., Fonseka, C.Y., McCole, R.B., Li, J.B., Senaratne, T.N., Williams, B.R., Rouillard, J.M., and Wu, C.T. 2012. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Natl. Acad. Sci. U.S.A. 109:21301‐21306.
  Bienko, M., Crosetto, N., Teytelman, L., Klemm, S., Itzkovitz, S., and van Oudenaarden, A. 2013. A versatile genome‐scale PCR‐based pipeline for high‐definition DNA FISH. Nat. Methods 10:122‐124.
  Boyle, S., Rodesch, M.J., Halvensleben, H.A., Jeddeloh, J.A., and Bickmore, W.A. 2011. Fluorescence in situ hybridization with high‐complexity repeat‐free oligonucleotide probes generated by massively parallel synthesis. Chromosome Res. 19:901‐909.
  Cremer, M., Gasser, F., Lanctôt, C., Müller, S., Neusser, M., Zinner, R., Solovei, I., and Cremer, T. 2008. Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes. Methods Mol. Biol. 463:205‐239.
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  Levsky, J.M. and Singer, R.H. 2003. Fluorescence in situ hybridization: Past, present and future. J. Cell. Sci. 116:2833‐2838.
  Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D.C. 1988. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80:224‐234.
  Martinez, A.M., Colomb, S., Déjardin J., Bantignies, F., and Cavalli, G. 2006. Polycomb group‐dependent Cyclin A repression in Drosophila. Genes Dev. 20:501‐513.
  Navin, N., Grubor, V., Hicks, J., Leibu, E., Thomas, E., Troge, J., Riggs, M., Lundin, P., Månér, S., Sebat, J., Zetterberg, A., and Wigler, M. 2006. PROBER: Oligonucleotide FISH probe design software. Bioinformatics 22:2437‐2438.
  O'Keefe, C.L., Warburton, P.E., and Matera, A.G. 1996. Oligonucleotide probes for alpha satellite DNA variants can distinguish homologous chromosomes by FISH. Human Mol. Genet. 5:1793‐1799.
  Pardue, M.L. and Gall, J.G. 1969. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl Acad. Sci. U.S.A. 64:600‐604.
  Pinkel, D., Straume, T., and Gray, J.W. 1986. Cytogenetic analysis using quantitative, high‐sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. U.S.A. 83:2934‐2938.
  Player, A.N., Shen, L.P., Kenny, D., Antao, V.P., and Kolberg, J.A. 2001. Single‐copy gene detection using branched DNA (bDNA) in situ hybridization. J. Histochem. Cytochem. 49:603‐612.
  Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A., and Tyagi, S. 2008. Imaging individual mRNA molecules using multiple singly‐labeled probes. Nat. Methods 5:877‐879.
  Silahtaroglu, A.N., Tommerup, N., and Vissing, H. 2003. FISHing with locked nucleic acids (LNA): Evaluation of different LNA/DNA mixmers. Mol. Cell. Probes 17:165‐169.
  Volpi E.V., and Bridger, J.M. 2008. FISH glossary: An overview of the fluorescence in situ hybridization technique. Biotechniques 45:385‐390.
  Yamada, N.A., Rector, L.S., Tsang, P., Carr, E., Scheffer, A., Sederberg, M.C., Aston, M.E., Ach, R.A., Taslenko, A., Sampras, N., Peter, B., Bruhn, L., and Brothman, A.R. 2011. Visualization of fine‐scale genomic structure by oligonucleotide‐based high‐resolution FISH. Cytogenet. Genome Res. 132:248‐254.
Key Reference
  Beliveau et al., 2012. See above.
  Introduces the Oligopaint FISH method and demonstrates its efficacy in tissue culture cells and tissue specimens.
Internet Resources
  http://genetics.med.harvard.edu/oligopaints/
  The Oligopaints Web site contains additional protocols, information about the location of probe sequences in several eukaryotic organisms, and scripts + documentation to assist users in the computational design of Oligopaint probes.
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