Molecular Combing

Chiara Conti1, Sandrine Caburet1, Catherine Schurra1, Aaron Bensimon1

1 Institut Pasteur, Paris
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.10
DOI:  10.1002/0471142956.cy0810s16
Online Posting Date:  May, 2001
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Abstract

This unit describes an important advance in fiber‐FISH technology called molecular combing, in which single DNA molecules are bound by one or both ends to a surface and stretched in a uniform and parallel manner by a receding meniscus. This technique is gentle on the molecules, rapid, and easy to perform. Reliable, quantitative information for genome‐wide studies can be obtained without the need for other techniques and a large number of accurate measurements can be made in a single experiment. The authors provide detailed protocols for basic molecular combing, high‐resolution physical mapping, and gene‐dosage approaches as well as support protocols outlining surface preparation, DNA solution preparation, and probe labeling.

     
 
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Table of Contents

  • Basic Protocol 1: Dynamic Molecular Combing of DNA
  • Alternate Protocol 1: High‐Resolution Genomic Mapping by Fluorescence In Situ Hybridization (Fish) on Combed DNA
  • Alternate Protocol 2: Gene Dosage by Molecular Combing
  • Support Protocol 1: Cleaning and Silanization of Glass Surfaces
  • Support Protocol 2: Preparation of DNA Solution
  • Support Protocol 3: Probe Labeling
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Dynamic Molecular Combing of DNA

  Materials
  • YOYO‐1‐stained DNA solution, pH 5.5 (see protocol 5 for DNA preparation)
  • Superglue
  • Combing apparatus with 1.5‐ml Teflon reservoirs (Pasteur Institute)
  • Silanized glass surfaces (see protocol 4)
  • Inverted epifluorescence microscope
  • Optical filter suitable for green fluorescence (FITC)
  • Video camera (e.g., SIT 68, Dage‐MTI)
  • Video recording device (Panasonic)
  • 60°C incubator
  • Microscope slides

Alternate Protocol 1: High‐Resolution Genomic Mapping by Fluorescence In Situ Hybridization (Fish) on Combed DNA

  • Probe cosmids, labeled with two (biotin and digoxigenin) or three (biotin, digoxigenin, and FITC) different haptens
  • 10 mg/ml herring sperm DNA (Table 8.10.1)
  • 1 mg/ml human cot‐1 DNA (Table 8.10.1)
  • 3 M sodium acetate, pH 5.2 (Table 8.10.1)
  • 70%, 90%, and 100% ethanol, cold
  • Hybridization buffer (Table 8.10.1)
  • Slides with combed DNA stored at −20°C (from protocol 1)
  • 50% and 70% (v/v) formamide in 2× SSC
  • Rubber cement
  • 2× SSC (Table 8.10.1)
  • Blocking solution (Table 8.10.1)
  • 0.05% (v/v) Tween 20 in 4× SSC
  • Avidin coupled to Texas Red (Av‐TR; Table 8.10.1)
  • Mouse anti‐digoxigenin coupled to FITC (mαdig‐FITC; Table 8.10.1)
  • Dilution buffer (DB; Table 8.10.1)
  • Biotinylated anti‐avidin (AAB; Table 8.10.1)
  • Donkey anti‐mouse coupled to FITC (dαm‐FITC; Table 8.10.1)
  • Mouse anti‐rabbit coupled to FITC (mαrb‐FITC; Table 8.10.1)
  • Rabbit anti‐FITC (F1; Table 8.10.1)
  • Anti‐rabbit coupled to FITC (F2; Table 8.10.1)
  • Avidin coupled to AMCA (Av‐AMCA; Table 8.10.1)
  • Mouse anti‐digoxigenin coupled to Texas Red (mαdig‐TR)
  • Anti‐mouse coupled to digoxigenin (αm‐dig)
  • 1× PBS ( appendix 2A)
  • Vectashield (Vector Labs)
  • Transparent nail polish
  • Speedvac evaporator (Savant)
  • 76°C water bath
  • Humid chamber (any tightly closing box protecting the slide from light and desiccation)
  • Coverslips
  • 37°C incubator
  • Epifluorescence microscope equipped with a CCD video camera
  • Image acquisition software (CartographiX, see )
  • Specific software for data analysis (e.g., CartographiX, see )
    Table 8.0.1   Additional Materials (also see protocol 1)   Additional MaterialsReagents and Solutions

    Name Stock solution Storage temperature a Concentration in reaction Reaction buffer Temperature a Pre‐treatment
    Anti‐avidin made in goat, coupled to biotin (AAB) 0.5 mg −20°C 1:50 (v/v) DB (see recipe) 37°C
    ABS Powder 4°C 6% 1× PBS RT Made fresh
    β‐agarase 1 1000 U/ml −20°C 8 U/ml 10× NE buffer 40°C
    β‐agarase 1 buffer 10× −20°C TE 40°C
    Agarose Powder RT 1.3% 1× TBE RT
    Anti‐digoxigenin made in mouse, coupled to FITC (mαdig‐FITC) 1.6 mg/ml −20°C 1:50 (v/v) DB (see recipe) 37°C
    Anti‐digoxigenin‐AP Fab fragments 150 U/200 µl +4°C 75 U/10 ml 6% BSA 37°C
    Avidin coupled to 7‐amino‐4‐methylcoumarin‐3‐acetic acid (Av‐AMCA) 5 mg/ml −20°C 1:10 (v/v) DB (see recipe) 37°C
    Anti‐FITC made in rabbit (F1) 0.4 mg/ml +4°C 1:100 (v/v) or 1:400 (v/v) DB (see recipe) 37°C
    Anti‐rabbit made in goat, coupled to FITC (F2) +4°C 1:100 (v/v) DB (see recipe) 37°C
    Anti‐mouse made in donkey, coupled to FITC (dαm‐FITC) 1.4 mg/ml −20°C 1:50 (v/v) DB (see recipe) 37°C
    Anti‐mouse made in sheep, coupled to digoxigenin (αm‐dig) 200 µg/ml −20°C 1:50 DB (see recipe) 37°C
    Anti‐rabbit made in mouse, coupled to FITC (mαrb‐FITC) 1.5 mg/ml −20°C 1:50 (v/v) DB (see recipe) 37°C
    AP‐DB buffer RT
    Argon RT RT
    Avidin coupled to Texas Red (Av‐TR) 2 mg/ml −20°C 1/50 v/v DB (see recipe) 37°C
    Bromochloroindolyl phosphate (BCIP) −20°C TP detection buffer (see recipe) RT Made fresh
    Bioprime DNA labeling system See manufacturer's instructions −20°C See manufacturer's instructions 37°C
    Blocking solution Powder RT in powder, −20°C when diluted 1.5% 0.05% Tween 20 in 4× SSC, pH 7.2 37°C 2 hr at 68°C
    BLUGENE nonradioactive nucleic acid detection system See manufacturer's instructions +4°C See manufacturer's instructions 6% BSA 37°C
    dNTP 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP −20°C 1:10 (v/v) Water 37°C
    DB (dilution buffer) 1:2 (v/v) 0.05% Tween 20 in 4× SSC/blocking solution Freshly made 37°C
    Digoxigenin‐11‐dUTP 1 nmol/µl −20°C 2 nmol/50 µl Water 37°C
    Dodecyl lithium sulfate (DLS) 100 mM NaCl, 50 mM EDTA, 100 mM Tris, 1% dodecyl lithium sulfate, water, pH 8 Freshly made
    EDTA/sarcosyl/proteinase K buffer (ESP) 4 ml proteinase K (10 mg/ml), 1% N‐lauroylsarcosine, 10 mM Tris Freshly made 0.5 M EDTA, pH 8 50°C 30 min at 50°C
    Ethidium bromide 10 mg/ml +4°C 0.5% 60 µl in 300 ml water RT
    Fluorescein‐11‐dUTP −20°C See manufacturer's instructions Water 37°C
    Herring DNA 10 mg/ml −20°C 10 µg per reaction Hybridization buffer (see recipe) 37°C
    Human cot‐1 DNA 1 mg/ml −20°C 5× quantity of probes Hybridization buffer (see recipe) 37°C
    Hybridization buffer 12.5 ml deionized formamide, 2.5 ml Tween 20, 6.25 ml dextran sulfate, 2.5 ml 20× SSC, up to 25 ml with water −20°C 20 µl per surface 37°C
    Labeled probes −20°C Cosmids: 400 ng on yeast, 700 ng on human BAC: 1 µg Hybridization buffer (see recipe) 37°C Precipitated in 100% ethanol and high salt concentration
    Lithium chloride (LiCl) 4.4 M RT 1:10 (v/v) Ethanol −70°C
    LMP agarose 1% +4°C 0.5% 1× TBE 42°C
    β‐mercaptoethanol 14.3 M RT 0.126% SCE (see recipe) 37°C
    2‐[N‐morpholino]ethane sulfonic acid (MES) 0.5 M, pH 5.5 Powder, at RT Water +4°C
    Sodium acetate 3 M, pH 5.2 RT 1:10 (v/v) Mix for hybridization solution
    Nitroblue tetrazolinium (NBT) −20°C TP detection buffer 37°C
    7‐Octenyltrichlorosilane (silane) 100% RT 100% RT
    OrangeG Powder +4°C 0.35% (w/v) 30% (w/v) glycerol in water −20°C
    PBS 10×, pH 7.4 RT Water
    SSC 20× RT 5×, 2×, 4× 0.05% Tween 20 Water RT
    Sorbitol/citrate/EDTA buffer (SCE) 1 M sorbitol, 10 mM EDTA, 100 mM sodium citrate, up to 100 ml water, pH 8.5 Freshly made Stock 37°C
    TBE 20× RT 0.5×, 1× Water RT
    TE 10 ml 1 M Tris, 2 ml 0.5 M EDTA, up to 1 liter with distilled water autoclave RT Water
    T 40E 2 4 ml 1 M Tris, 0.4 ml 0.5 M EDTA, up to 100 ml with distilled water autoclave RT Water RT
    Phenylmethylsulfonyl fluoride solution (PMSF) 40 mg/ml in isopropanol RT 1:1000 (v/v) TE (see recipe) 50°C
    AP‐DB buffer 100 mM Tris⋅Cl, 100 mM NaCl, 50 mM MgCl 2 RT
    UltraPure agarose 1.3% RT 1× TBE
    YOYO‐1 1 mM +4°C 1 µl in 150 µl T 40E 2 (see recipe) RT
    Zymolyase 20T Powder +4°C in powder, −20°C in solution 1 U/µl 50 mM Tris⋅Cl, pH 7.4, 1 mM EDTA, pH 8, 50% glycerol, sterilize
    Zymolyase buffer 0.126% (v/v) SCE buffer, 12.6 µl β‐mercaptoethanol in 100 ml final 10 U/ml zymolyase 20T RT 37°C

     aAbbreviation: RT,room temperature.

Alternate Protocol 2: Gene Dosage by Molecular Combing

  • Probes specific for a control region and the target region, labeled with two different haptens
  • Additional reagents and equipment for preparation of molecular combing (see protocol 1), DNA combing solution (see protocol 5), and probe labeling (see protocol 6)

Support Protocol 1: Cleaning and Silanization of Glass Surfaces

  Materials
  • Acetone
  • Pyrolyzed water
  • 7‐Octenyltrichlorosilane (silane; Table 8.10.1)
  • Chloroform
  • Ethanol
  • 22 × 22–mm coverslips (ESCO)
  • Ceramic coverslip holders
  • Argon and oxygen supplies
  • Flow metering units
  • Gas hydration device
  • UV lamp covered with quartz
  • Connecting tubing and appropriate valves
  • Closed treatment cell (1‐liter volume)
  • Small Erlenmeyer (silane reservoir)
  • 100‐µl glass syringe (Hamilton)

Support Protocol 2: Preparation of DNA Solution

  Materials
  • Desired cells
  • 1× PBS, pH 7.4, ice cold
  • Low melting point agarose (LMP agarose; FMC Bioproducts)
  • EDTA/sarcosyl/proteinase K buffer (ESP solution; Table 8.10.1)
  • TE/phenylmethylsulfonyl fluoride solution (TE/PMSF; Table 8.10.1)
  • 0.5 M EDTA, pH 8.5 ( appendix 2A)
  • Sorbitol/citrate/EDTA buffer (SCE; Table 8.10.1)
  • Zymolyase 20T and buffer (Table 8.10.1)
  • 14.3 M β‐mercaptoethanol
  • Dodecyl lithium sulfate buffer (DLS; Table 8.10.1)
  • 1× TE (Table 8.10.1)
  • 1.3% (w/v) agarose gel in 0.5× TBE
  • 0.5× TBE (Table 8.10.1)
  • 1 mM YOYO‐1 in T 40 E 2 (Table 8.10.1)
  • 10× β‐agarase 1 buffer (NEB)
  • β‐agarase 1
  • 0.5 M 2‐[N‐morpholino]ethane sulfonic acid (MES; Table 8.10.1), pH 5.5
  • 37°C and 50°C waterbaths
  • 100‐µl pulsefield block molds (BioRad)
  • Rotating wheel
  • 15‐ml tubes
  • 2‐ml round‐bottom Eppendorf tubes

Support Protocol 3: Probe Labeling

  Materials
  • Bioprime DNA labeling system (Life Technologies), including:
  •  2.5× random primers
  •  Klenow fragment
  •  10× dNTP mix (with biotin‐16‐dUTP included)
  • Extracted DNA (cosmids, BAC, YAC)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • 3 M sodium acetate, pH 5.2 (Table 8.10.1)
  • 100% ethanol
  • 1× TE (Table 8.10.1)
  • Digoxigenin‐11‐dUTP
  • 4.4 M lithium chloride (LiCl; Table 8.10.1)
  • Fluorescein‐11‐dUTP (FluoroGreen, Amersham)
  • QIAquick PCR purification kit (QIAGEN)
  • 100 mM dATP Li‐salt, 100 mM dCTP Li‐salt, 100 mM dGTP Li‐salt, 100 mM dTTP Li‐salt in distilled water
  • UltraPure agarose (Life Technologies; also see Table 8.10.1)
  • 1× TBE buffer (Table 8.10.1)
  • OrangeG (Sigma; Table 8.10.1)
  • 0.25 µg/µl λ DNA
  • 10 mg/ml ethidium bromide (Table 8.10.1)
  • 5 ng/µl dig‐labeled control DNA
  • BLUGENE nonradioactive nucleic acid detection system (Life Technologies)
  • Hybond membranes (Amersham Pharmacia Biotech)
  • 6% BSA (Table 8.10.1)
  • 0.4 N NaOH
  • 5× SSC (Table 8.10.1)
  • 1× PBS
  • Anti‐digoxigenin‐AP Fab fragments (Table 8.10.1)
  • AP‐DB buffer (Table 8.10.1)
  • Bromochloroindolyl phosphate (BCIP; Table 8.10.1)
  • Nitroblue tetrazolinium (NBT; Table 8.10.1)
  • Speedvac evaporator
  • Electrophoresis apparatus
  • UV transilluminator
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Figures

Videos

Literature Cited

Literature Cited
   Allemand, J.F., Bensimon, D., Jullien, L., Bensimon, A., and Croquette, V. 1997. pH‐dependent specific binding and combing of DNA. Biophys. J. 73:2064‐2070.
   Bensimon, A., Simon, A., Chiffaudel, A., Croquette, V., Heslot, F., and Bensimon, D. 1994. Alignment and sensitive detection of DNA by a moving interface. Science 265:2096‐2098.
   Bensimon, D., Simon, A., Croquette, V., and Bensimon, A. 1995. Stretching DNA with a receding meniscus: Experiments and models. Phys. Rev. Lett. 76:4754‐4795.
   Florijn, R.J., Bonden, L.A., Vrolijk, H., Wiegant, J., Vaandrager, J.W., Baas, F., den Dunnen, J.T., Tanke, H.J., van Ommen, G.J., and Raap, A.K. 1995. High‐resolution DNA fiber‐FISH for genomic DNA mapping and colour bar‐coding of large genes. Hum. Mol. Genet. 4:831‐836.
   Herrick, J. and Bensimon, A. 1999. Single molecule analysis of DNA replication. Biochimie 81:859‐871.
   Herrick, J., Stanislawski, P., Hyrien, O., and Bensimon, A. 2000a. Replication fork density increases during DNA synthesis in X. laevis egg extracts. J. Mol. Biol. 300:1133‐1142.
   Herrick, J., Michalet, X., Conti, C., Schurra, C., and Bensimon, A. 2000b. Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA [published erratum appears in Proc. Natl. Acad. Sci. U.S.A. 97:4410. Proc. Natl. Acad. Sci. U.S.A. 97:222‐227.
   Michalet, X., Ekong, R., Fougerousse, F., Rousseaux, S., Schurra, C., Hornigold, N., van Slegtenhorst, M., Wolfe, J., Povery, S., Beckmann, J.S., and Bensimon, A. 1997. Dynamic molecular combing: Stretching the whole human genome for high‐resolution studies. Science 277:1518‐1523.
   Strick, T.R., Allemand, J.F., Bensimon, D., Bensimon, A., and Croquette, V. 1996. The elasticity of a single supercoiled DNA molecule. Science 271:1835‐1837.
   Weier, H.U., Wang, M., Mullikin, J.C., Zhu, Y., Cheng, J.F., Greulich, K.M., Bensimon, A., and Gray, J.W. 1995. Quantitative DNA fiber mapping. Hum. Mol. Genet. 4:1903‐1910.
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