User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

High‐Resolution FISH Analysis

Henry H.Q. Heng¹, Bradford Windle², Lap‐Chee Tsui³

¹Center for Molecular Medicine and Genetics Wayne State University School of Medicine, Detroit, Michigan
²Virginia Commonwealth University, Richmond, Virginia
³University of Hong Kong, Hong Kong

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 4.5

DOI:

10.1002/0471142905.hg0405s44

Online Posting Date:

February, 2005

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

Abstract

Map order, orientation, and gap or overlap distance of closely linked DNA probes may be determined using fluorescent hybridization to decondensed DNA. The linear arrangement of released chromatin fibers not only simplifies the task of gene ordering, but also provides higher resolution with probes separated by greater distances than can be achieved in FISH with intact interphase nuclei. The Basic Protocol 1 of this unit describes an alkaline lysis procedure for generating free chromatin from cultured cells for FISH analysis. A support protocol describes an empirical approach to optimize conditions for preparation of free chromatin. An Alternate Protocol 1 provides a method for producing free chromatin from cultured lymphocytes with drug treatment. The Basic Protocol 2, high-resolution FISH mapping with free chromatin, is a modification of the method used for FISH mapping of interphase nuclei.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Unit Introduction
Basic Protocol 1: Preparation of Chromatin Fiber with Alkaline Buffer
Support Protocol 1: Optimization of Chromatin Fiber Preparation
Alternate Protocol 1: Preparation of Chromatin Fibers from Lymphocytes by Drug Treatment
Preparation of DNA Fibers
Basic Protocol 2: Preparation of DNA Fibers Using SDS Lysis and Gravity
Alternate Protocol 2: Preparation of DNA Fibers Using Alkaline Treatment Plus Mechanical Pulling
Basic Protocol 3: Fish Detection with Stretched Chromatin and DNA Fibers
Support Protocol 2: Biotin and Digoxigenin Labeling of Fish Probes
Reagents and Solutions
Commentary
Literature Cited
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Chromatin Fiber with Alkaline Buffer

Materials

Fibroblast culture or 10 ml fresh human peripheral or cord blood
Fibroblast or lymphocyte culture medium (see recipe; with and without serum for fibroblast medium)
Trypsin/EDTA solution (Invitrogen)
Alkaline buffer (see recipe)
Fixative: 3:1 (v/v) methanol/glacial acetic acid (prepare fresh)

60-mm tissue culture plates
25-cm² tissue culture flasks
15-ml screw-cap polystyrene tubes
IEC HN-S centrifuge and 958 (or equivalent) rotor
Microscope slides, chilled 5 to 10 min on ice
Phase-contrast microscope
Slide box

Additional reagents and equipment for tissue culture and trypsinization of cells (appendix 3G)

Support Protocol 1: Optimization of Chromatin Fiber Preparation

Additional Materials (also see Basic Protocol 1)

4% Giemsa stain: 2 ml Giemsa stain (Fisher)/48 ml Gurr, pH 6.8 (BDH), prepared fresh

Alternate Protocol 1: Preparation of Chromatin Fibers from Lymphocytes by Drug Treatment

Additional Materials (also see Basic Protocol 1)

3 ml fresh human peripheral or cord blood
5 mg/ml m-AMSA (see recipe) or 10 mg/ml ethidium bromide (appendix 2D)
0.4% (w/v) KCl

CAUTION: m-AMSA and ethidium bromide are mutagens and should be handled with care.

Basic Protocol 2: Preparation of DNA Fibers Using SDS Lysis and Gravity

Materials

Cultured cells
Phosphate-buffered saline (PBS; appendix 2D)
Lysis buffer (see recipe)
Fixative: 3:1 (v/v) methanol/glacial acetic acid (prepare fresh)
N₂ gas

25 × 75 × 1–mm glass microscope slides
Moist chamber (Fig. 4.3.3)
Heat-sealable bags
Drierite, 8-mesh (Fisher)

Alternate Protocol 2: Preparation of DNA Fibers Using Alkaline Treatment Plus Mechanical Pulling

Materials

Cultured cells
0.4% (w/v) KCl
Fixative: 3:1 (v/v) methanol/glacial acetic acid (prepare fresh)
Phosphate-buffered saline (PBS; appendix 2A)
Alkaline solution: 5:2 (v/v) 0.07 M NaOH/100% ethanol (prepare fresh)
Methanol
70%, 95%, and 100% ethanol

15-ml centrifuge tubes
IEC HN-S centrifuge and 958 (or equivalent) rotor
25 × 75 × 1–mm glass microscope slides
Coverslips

Basic Protocol 3: Fish Detection with Stretched Chromatin and DNA Fibers

Materials

Glass slides containing streams of stretched DNA or stretched chromatin fiber (see Basic Protocol 1 or 2, or Alternate Protocol 2)
100 µg/ml RNase: dilute 2 mg/ml RNase stock (DNase-free; appendix 2D) in 2× SSC; prepare fresh
4× and 2× SSC (appendix 2D)
70% (v/v) deionized formamide/2× SSC, 70°C
70% (v/v) ethanol, ice-cold
100% and 90% (v/v) ethanol, room temperature
Labeled probes (see Support Protocol 2)
Sonicated genomic DNA (100 to 1000 bp; see recipe) or C₀t₁ DNA (e.g., COT-1 DNA, Invitrogen)
Hybridization buffer (see recipe)
Rubber cement
50% (v/v) nondeionized formamide/2× SSC
Preavidin block solution (see recipe)
5 µg/ml fluorescein-avidin DCS (Vector) in 4× SSC/1% (w/v) BSA
4× SSC/0.1% (v/v) Triton X-100
PN buffer (see recipe)
NGS/PN solution: 4% (v/v) normal goat serum (NGS, Vector) in PN buffer (store 1-ml aliquots at 4°C)
5 µg/ml biotinylated anti-avidin D antibody (Vector) in NGS/PN solution
25 µg/ml mouse anti-digoxigenin antibody (Roche) in NGS/PN solution
25 µg/ml digoxigenin-labeled polyvalent anti-mouse Ig F(ab¢)₂ fragment (Roche) in NGS/PN solution
25 µg/ml rhodamine-conjugated anti-digoxigenin Fab fragment (Roche) in NGS/PN solution
Vectashield antifade mounting medium (Vector)

22 × 40–mm glass coverslips, no. 1
Polyethylene Coplin jars
Moist chamber (Fig. 4.3.3)
45° and 75°C water baths
Rubber cement
Epifluorescence microscope (unit 4.4) with triple-band-pass filter (or set of single-band-pass filters for FITC and rhodamine) and 100× oil objective
CCD camera (e.g., Photometrics CoolSNAP)
Image analysis software (e.g., Photometrics RS Image)

Support Protocol 2: Biotin and Digoxigenin Labeling of Fish Probes

Materials

Probe DNA to be labeled
Nick translation DNA labeling kit, e.g., BioNick (Invitrogen) or Boehringer DIG nick translation mix (Roche Applied Sciences); alternatively, prepare nick translation mix in-house (see recipe)
Stop buffer: 0.5 M EDTA, pH 8.0 (appendix 2D)
TE buffer, pH 7.5 (appendix 2D)
Sonicated salmon sperm DNA (Invitrogen; see unit 4.3 for sonication)
3 M sodium acetate (appendix 2D)
70% and 100% ethanol

15°C water bath
Nick column (Amersham Pharmacia Biotech)

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 4.5.1

FISH detection on extended DNA fiber. (A) The fiber FISH image of two overlapping probes. Two BAC clones were labeled and detected with green and red color respectively. The overlapping portion displays a blended yellow color. This allows detailed analysis of the overlapped region. (B) A gap between two known probes that are colored green and red. Since the DNA fiber is linearized and the size of both probes are known, the gap can be quantitatively measured using the length of the known probes as a measuring gauge. (C) An orientation analysis utilizing fiber FISH of three BAC probes labeled and detected as red, green, and yellow colors respectively. The relative positions as well as the relative size can be easily visualized and measured. (D) Mapping of multiple replication initiation sites within a centromeric repeated DNA sequence region. CHO cells were pulse-labeled with BrdU. DNA was stretched and the sites of replication initiation on DNA strands were detected with fluorescein (green) using anti-BrdU antibodies. Fiber FISH was used to detect the location of DNA strands containing centromeric repeats of the TTAGGG sequence, labeled with rhodamine (red). The DNA strand shown contains multiple yet separate regions of TTAGGG repeats that span ~3 to 5 kb each. Interspersed between these repeat regions are ~3 to 5 kb of unique DNA sequences, which are where the many replication initiation sites are located.

View Image
Figure 4.5.2

FISH mapping with chromatin fibers. (A) Ordering repetitive elements in the centromeric region of human chromosome 15 by chromatin fiber FISH mapping. Three different repetitive probes—(a) labeled with biotin only, (b) labeled with 50% biotin/50% digoxigenin, and (c) labeled with digoxigenin alone—show green, yellow, and red color, respectively. The order of the three probe-hybridizing regions is a-b-c. In contrast, in a nearby interphase nucleus, the signals for probe b and c are intermixed and the order cannot be determined. The probe-hybridizing regions a, b, and c span a 2-Mb region. (B) An example of ordering cosmid probes by chromatin fiber FISH mapping. The panel shows the chromatin fibers used in the mapping, stained with DAPI. The insert panel shows the order of three cosmids (probes e, f and g) in the region of human chromosome 7q22, represented by green, yellow, and red color signals, respectively.

View Image
Figure 4.5.3

Diagram of DNA condensation and organization in chromosome preparations for FISH. The extent of DNA condensation and level of organization are depicted for a metaphase chromosome, an interphase nucleus, chromatin fibers, and a naked (protein-depleted) DNA fiber. In both the metaphase chromosome and interphase nucleus, the particular region of the chromosome is condensed and arranged three-dimensionally. The 30-nm chromatin fibers are released in a bundle from the interphase nucleus. The target chromatin exists in a less condensed and more linearized state. The 30-nm chromatin can be further decondensed to form naked DNA fiber by depleting it of proteins.

View Image

Literature Cited

Literature Cited
	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.
	Datson, N.A., Semina, E., van Staalduinen, A.A., Dauwerse, H.G., Meershoek, E.J., Heus, J.J., Frants, R.R., den Dunnen, J.T., Murray, J.C., and van Ommen, G.J. 1996. Closing in on the Rieger syndrome gene on 4q25: Mapping translocation breakpoints within a 50-kb region. Am J Hum Genet. 59:1297-305.
	Dunham, I., Shimizu N., Roe, B.A., Chissoe, S., Hunt, A.R., Collins, J.E., Bruskiewich, R., Beare, D.M., Clamp, M., Smink, L.J., Ainscough, R., Almeida, J.P., Babbage, A., Bagguley, C., Bailey, J., Barlow, K., Bates, K.N., Beasley, O., Bird, C.P., Blakey, S., Bridgeman, A.M., Buck, D., Burgess, J., Burrill, W.D., O'Brien, K.P. 1999. The DNA sequence of human chromosome 22. Nature. 402:489-95.
	Fidlerova, H., Senger, G., Kost, M., Sanseau, P., and Sheer, D. 1994. Two simple procedures for releasing chromatin from routinely fixed cells for fluorescence in situ hybridization. Cytogenet. Cell Genet. 65:203-205.
	Fransz, P.F., Alonso-Blanco, C. M., Liharska, T.B., Peeters, A.J.M., Zabel, P., and de Jong, J.H. 1996. High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres, Plant J. 9:421-430.
	Gervasini, C., Bentivegna, A., Venturin, M., Corrado, L., Larizza, L., and Riva, P. 2002. Tandem duplication of the NF1 gene detected by high-resolution FISH in the 17q11.2 region, Hum. Genet. 111:465-467.
	Haaf, T. and Ward, D.C. 1994. Structural analysis of -satellite DNA and centromere proteins using extended chromatin and chromosomes. Hum. Mol. Genet. 3:697-709.
	Haaf, T. 1996. High-resolution analysis of DNA replication in released chromatin fibers containing 5-bromodeoxyuridine, Biotechniques 21:1050-1054.
	Heiskanen, M., Karhu, R., Hellsten, E., Peltonen, L., Kallioniemi, O.P., and Palotie, A. 1994. High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells. BioTechniqes. 17:928-934.
	Heng, H.H.Q., Squire, J., and Tsui, L.-C. 1992. High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc. Natl. Acad. Sci. U.S.A. 89:9509-9513.
	Heng, H.H.Q. and Tsui, L.-C. 1993. Modes of DAPI banding and simultaneous in situ hybridization. Chromosoma 102:325-332.
	Heng, H.H.Q., Tsui, L-C., and Moens, P.B. 1994a. Organization of heterologous DNA inserts on the mouse meiotic chromosome core. Chromosoma 103:401-407.
	Heng, H.H.Q., Xiao, H., Shi, X.-M., Greenblatt, J., and Tsui, L.-C. 1994b. Genes encoding general initiation factors for RNA polymerase II transcription are dispersed in the human genome. Hum. Mol. Genetics. 3:61-64.
	Heng, H.H.Q., Chamberlain, J.W., Shi, X.-M., Spyropoulos, B., Tsui, L.-C., Moens, P.B. 1996. Regulation of meiotic chromatin loop size by chromosomal position. Proc. Natl. Acad. Sci. U.S.A. 93:2795-2800.
	Heng, H.H.Q., Spyropuulos, B., Moens, P.B. 1997. FISH technology in chromosome and genome research. Bioessays 19:75-84.
	Heng, H.H.Q. and Tsui, L.-C. 1998. High resolution free chromatin/DNA fiber fluorescent in situ hybridization. J. Chromatog. 806:219-229.
	Heng, H.H.Q. 2002. High resolution FISH mapping using chromatin and DNA fiber. In FISH: A Practical Approach (B.G. Beatty, S. Mai and J.A. Squire, eds.) p. 77-92. Oxford University Press, New York.
	Heng, H.H.Q., Goetze, S., Ye, C.J., Liu, G., Stevens, J.B., Bremer, S.W., Bode, J., Wykes, SM., and Krawetz, SA. 2004. Chromatin loops are selectively anchored using scaffold/matrix attachment regions. J. Cell. Sci. 117:999-1008.
	Horelli-Kuitunen, N., Aaltonen, J., Yaspo, M.L., Eeva, M., Wessman, M., Peltonen, L., and Palotie, A. 1999. Mapping ESTs by fiber-FISH. Genome Res. 9:62-71.
	Houseal, T.W., Dackowski, W.R., Landes, G.M., and Klinger, K.W. 1994. High resolution mapping of overlapping cosmids by fluorescence in situ hybridization. Cytometry 15:193-198.
	Iafrate, A.J., Feuk, L., Rivera, M.N., Listewnik, M.L., Donahoe, P.K., Qi, Y., Scherer, S.W., and Lee, C. 2004. Detection of large-scale variation in the human genome. Nat. Genet. 36:949-951.
	Inoue, K., Osaka, H., Imaizumi, K., Nezu, A., Takanashi. J., Arij, J., Murayama, K., Ono, J., Kikawa, Y., Mito, T., Shaffer, L., and Lupski, J. 1999. Proteolipid protein gene duplications causing Pelizaeus-Merzbacher Disease: Molecular mechanism and phenotypic manifestations. Ann. Neurol. 45:624-632.
	Lestou, V.S., Strehl, S., Lion, T., Gadner, H., and Ambros, P.F. 1996. High-resolution FISH of the entire integrated Epstein-Barr virus genome on extended human DNA. Cytogenet. Cell. Genet. 74:211-217.
	Moens, P. and Pearlman, R.E. 1990. In situ DNA mapping with surface-spread mouse pachytene chromosomes. Cytogenet. Cell Genet. 53:219-220.
	Norio, P. and Schildkraut, C. 2001. Visualization of DNA replication on individual Epstein-Barr virus episomes. Science 294:2361-2364.
	Parra, I. and Windle, B. 1993. High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature Genet. 5:17-21.
	Pasero, P., Bensimon, A., and Schwob, E. 2002. Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus. Genes Dev. 16:2479-2484.
	Raap, A.K., van de Corput, M.P., Vervenne, R.A., van Gijlswijk, R. P., Tanke, H.J., and Wiegant, J. 1995. Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum. Mol. Genet. 4:529-534.
	Ried, T., Baldini, A., Rand, T.C., and Ward, D.C. 1992. Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. U.S.A. 89:1388-1392.
	Rottger, S., Yen, P.H., and Schempp, W. 2002. A fiber-FISH contig spanning the non-recombining region of the human Y chromosome. Chromosome Res. 10:621-635.
	Trask, B., Pinkel, D., and van den Engh, G. 1989. The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics 5:710-717.
	Trower, M.K., Orton, S.M., Purvis, I.J., Sanseau, P., Riley, J., Christodoulou, C., Burt, D., See, C.G., Elgar, G., Sherrington, R., Rogaev, E.I., St George-Hyslop, P., Brenner, S., and Dykes, C.W. 1996. Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus). Proc. Natl. Acad. Sci. U.S.A. 93:1366-1369.
	Tsuchiya, D. and Taga, M. 2001. Application of fibre-FISH (fluorescence in situ hybridization) to filamentous fungi: Visualization of the rRNA gene cluster of the ascomycete Cochliobolus heterostrophus. Microbiology. 147:1183-1187.
	van Ommen, G.J., Breuning, M.H., and Raap, A.K. 1995. FISH in genome research and molecular diagnostics. Curr. Opin. Genet. Dev. 5:304-308.
	Vesa, J., Hellsten, E., Verkruyse, L.A., Camp, L.A., Rapola, J., Santavuori, P., Hofmann, S.L., and Peltonen, L. 1995. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature. 376:584-587.
	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.
	Wiegant, J., Kalle, W.J., Mullenders, L.H.F., Brookes, S., Hoovers, J.M.N., Van Ommen, G.J.B., and Raap, A.K. 1992. High-resolution in situ hybridization using DNA halo preparations. Hum. Molec. Genet. 1:587-591.
	Windle, B., Silvas, E., and Parra, I. 1995. High resolution microscopic mapping of DNA using multicolor fluorescent hybridization. Electrophoresis 16:273-278.
	Windle, B., Parra, I., and Silvas, E. 1999. Analysis of structure and function by high-resolution visual mapping of extended DNA. In Introduction to Fluorescence In Situ Hybridization (M. Andreeff and D. Pinkel, eds.) pp. 119-132. John Wiley & Sons, New York.
Key References
	Heng et al., 1992. See above.
The first paper introducing the concept of high- resolution fiber FISH.
	Parra and Windle, 1993. See above.
Shows a variety of data generated with the DNA fiber mapping protocol