27

cytogefzetics

Using fluorescencein situ ybriclization ---i) [FISH) rn genomemapping Julie R. Korenberg, Teresa Yang-Feng, Rhona Schreck and Xiao Ning Chen Fluorescence in situ hybridization (FISH) provides one of the most effective and rapid approaches

for assigning and ordering DNA franments within single

eukaryotic chromosome bands. These techniques have wide applications not only for the mapping of the human genome and the genomes of other organisms, but also in clinical cytogenetics, somatic cell genetics, cancer diagnosis and gene

TCXIS i\cd. though rc‘ccnt dcvclopmcnts ill instrutiictit:ition have nllowcd the 11sc' of $4~3 which fluorcscr under infra-red” (‘Table 1). Althougil indirect svstcms arc more ividcly used bccnucc of the oppor&nity for signnl anipliiication using multipic rounds ofmribody or biotm-avidin application. tbcrc is n~ucb intcrcst ill the dcvcloprncnt of tlircc? kibcls that may dccrcisr hckgrwnd ;uid itiaensc tlrc $.plto-noise

mcio.

Probe hybtidizalion 0ptim;rl hybridization ccmditions &pcnd cln Chc mrturc of the probe and the tar.g+“. ~rirIiy. the ticllaturcd, I;~bclcd probe (size 1~11gc 700--500 bp) is Irybridizcd with dcll:lturcd-cltrolnosclmc prcpzatimn (ovcn~ight, ;It 37°C. in 50% forixmidc), !Tdlowcd by

gcntlc wnihing

to

rcnlow

uiiliyb~idizrd

or

Inis-

probe, foilowrd by hybrid dctcction. For gcnomic probes such as thocc cloned in lambd:~ phagc, cosmids or YACs, the c’:mturcd probe is prchybridized with csccss wdabeicd total DNA, O: DNA cmichcd in repent scc~~w~~cs (Cot I IjNA), to prcvent non-specific hybridization bctwccn probe ad matched

Table 1. Fluorescent reporter molecules Excitation maximum (nm) -._.-

AMCA”avidin

fluorescein avidin green Phycobiiierythrin R awidin Rhcdamine auidin Texas Red avidin 0

infra-red dyes

350

AMCAT-arw

450

495

450-570

ICY3. carboxymethyl indocyaninet

EL_

Emission maximum (nm) Color --____

550 595

650

4-methylcoumarin-3-acetic

Yellow

574

Red

575 615

Red Deep red Not visible

670

acid.

BIN

515

_-

--I

_.._. _...__..I_,__. --- - -------TiSTECHJANfFEB 1992 WQL 10)

28

cytogerzetics

Figure 3 Human chromosome telomere sequences are clearly shown using a human telomere probe. Centromeric heterochromatic regions of chromosomes 1, 9, 6 are shown by simultaneous hybridization of human satellite DNA sequence. ..

gcnoniic rcpcar scqucnccs 4 l’l.l 1and thcrcby rcducc the bnckgrollnd.

Figure 1 Human chromosonies shown with R (Reverse) banding generated with chromomycin A,/distamycin A. A cosmid for a human glutamate (Kainatej receptor maps to 5q33 as shown using the biotin-avidinFITC system (Ref. 17).

Figure 2 h chramazlme 4 ‘painting’ probe reveals multiple rearrangements of chromosome 4 in a hepatoma cell line. The mutation in this cell line was previously defined as associated with a chromosome 4 viral integration site. TIETECH JAN/FEB 1992 WOL 101

Signal detection: instrumen;~Goa Scandnrd photonlicroscopy, utilizing a high-qualiry fluorescence microscope ci+lipycd wi:lr a stnndxd Quiiiac!-inc film- set, is aI1q~i.m for the high resolution mapping of probes rmigi’i!ngiii six fiorll 1.5 kb to at Icast000 kb. Howcvcr, cxposurc tinlcs arc dcpclldcnt on the combination of film-type, probe lab&g sysmm, tluorophorcs, films, probe system and type of microscope used. The csposurc times requixcd for &tection ofwcnk signais (e.g. from single-copy, l-10 kb scqucnccs) using high-rctalution film may be ii] csccss of 300 s whcrcas shorter esposurc times of 10-4) s are sufficient for recording cosmid hybridizations oil lower resolution film. Although clearly visible through the microscope, fluorcsccnt signals from hybridized probe may bc d&cult to dictinguish from chrormosonlc banding patterns on the resulting blackand-white photographs. The USC ofvideo-ass&cd SIT (silicon intcnsificd t;Wget)caimcras greatly irnprovcs the ability to record a signai rapidly but their low resolution hnlits band assignment. The rcccnt dcvclopniclrt of the cooled CCD (chargc coupled device) canIcI.a ovcrconIcs thcsc problems and providcs an ;Lccurate lincnr record of the fluoresccncc along a cl~ro~nosomr~. With appropriate software for image analysis. the rcsolurion obtnincd with a CCD is cqu~valcnt to that ofthc light nlicroscopc. This sysstan, when coupled with niodcratc irmagc-analysis capnbilitics and the appropriate excitation and cniission filters, is capable of dctccring and recording rnul:iplc coiors and weak signals, displaying their inxxgcs supcrimposed on hii5h-1-[~solutiol1,banded chrormosolncs.

Figure 4 Achromosome 21 centromeric YAC (410 kb) reveals homologies with the centromeric regions of 2Iq, 14q and a noncentromeric region of 1 iq. (A) Standard photomicroscopy ailows defmition of chromosomq band position. (8) Image captured with the Hamamatsu Photon Counting Camera shows six specific sites in over 90% of cells. Probe DNAs donated by G. Stewart and D. Kurnit, Pasadena, USA.

Figure 5 A 220 hb human genomic fragment maps to a single site on chromosome 8 in all cells. (ii) image obtal!led with standard photomicroscopy. (B) image obt;ltned from Hamamatsu Camera (as in Fig. 4). Probe DNAs donated by M. Simon and H. Shizuya.

Confocnl nticrnscopy have ako been used Ruorcsccnt signals’.

and photon-counting catmms in dctccting and unaiysing thr

Multicolor FISH FISH is wcil suited for th(: simdtancous dctrction of more than one target scqucncc by using multicolor labcling. For csample, :hrec fktorophores cviitting in the green (FITC). the red (Tcm Red, rho&minc, TKITC. XRITC atid phycmxythtin) and the blue (7-amino--l-metl~yl-coum3ri~~-3-acctic acid

IAMCA]) may be used in combinnriort \vith cpifluorcsccncc microscopy and the nppm+W erzitation and ctnission filter cotnbination~. Multicolor irr sifrr hybridization is quite a rcccnt dcvcloptncnt 2nd ~nud~ resent-ch is still bcins dirrctcd at optimizing cotnpntThe iblc probe labels, fiitcrs and fluorochrcmcs. wcccssful use of tnultipfc probes depends also cm the abi!iv to obtain images of ditErcnt fluot-ochro~xs in rrgistmtion. Digitization and compmerizcd sror;lgc of inmgcs is one opticn. and it ~cnts iikciy thdi the vxolurion. image six 2nd spw~1 3F hnndling will

Figure 6 A human cDNA for complement factor 3 CC31 maps to cnromosome 19p13.3i. (A) Standard photomicroscopy and reverse banding (as in Fig. 11; 161 Hamamatsu Camera reveals clear signals on 90% of chromatids

be

able

to

match

photography

in tire

forcsccablc

fLlturc.

Ikccntly, 111~11tipk Lund-pass filter sets have been developed that permit the siniultancous viewing, with a low background, of a green FlTC signal and red (Terns Red) signal on DAPI-stained chromosomes. for dctcrmining registration This is CHIC option of multiple probe signals relative to chromosomal image.However, the narrow spectrum offluorescence ficqucncics imposed by these fiiters for dccrcasinp also decrcnsec the signal strcrrgth. background Thcrcforc, for the de:cction of small probes in tilt range of l-2 kb (kvhich would have rclativcly little .Issociatcd fluoresccncc), CCD iilstrumslI~~tiotl with the appropriate tiltcr combination is prcfcrabk.

Chromosome-banding techniqws and reference banding systems After FISH. chromosomes can 1~1:banded for idcntitkation, ;uld to localize probes to s~ccific chromosomal chromosome regions. The LISC of high-resolution preparations is csscntial for obtaining good rcfcrcncebanding patterns. Staining technic LICS (see H. Cook, pp. 23-X in this issue of TmT,ricxj fcjr Obt;liIiiil& banded chromosomes after FISH include: G (Gicmtn)/Q (Quinncrincj bunckng usilig l-locchst 33155 (Ref. i2), qmnacrinc #Jr I>AI’l; and R (ltcvcrsc) banding, by scqucntia! ss:aininy with fluox~ shromcs inclr!ding chromomycin A,/distamycin A (Ref. 13). Hoccilst 33258/propidium iodide (Retl 14) and DAPl/propidium iodide (Rc’f. 15). Chromosome banding cm also be achieved b>. direct hybridization of Alu LIHs (LIHs = Ll HONO r &tra:?s?:riptio::, *he . dctccric:; &