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A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA.

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A Sequence in

M13

Phage

Detects

Hypervariable

Minisatellites

in Human

and Animal DNA

GILBERT VASSART, MICHEL GEORGES, RITA MONSIEUR,

HUGUETrE BROCAS,

ANNE

SOPHIE

LEQUARRE, DANIEL CHRISTOPHE

The term "DNA fingerprint" has been used to describe the extensive restriction

fragment length polymorphism associated with hypervariable minisatellitespresent in

thehuman genome.Untilnow, it was necessary tohybridizeSouthern blots tospecific

probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns.The presentstudydescribes the surprising finding that the

insert-free,wild-type M13 bacteriophage detects hypervariableminisatellitesin human andin animalDNA, provided no competitor DNA is used during hybridization.Theeffective sequence in M13 was traced to two dustersof15-base pair repeats within the protein III gene of the bacteriophage. This unexpected use of M13 renders the DNA

fingerprinting technologymorereadilyavailable to molecular biology laboratories. E XTENSIVE RESTRICTION FRAGMENT

length polymorphism (RFLP)

asso-ciated with moderately repeated se-quences has recently been described in the

human genome (1-4). The hypervariable

nature of these "minisatellites" has been exploited as a meansof developinga

power-fiul

fingerprinting technique with

applica-tions inpaternitytesting,forensicmedicine, andmapping ofthe human genome (5, 6). The probes used in such determinations were originally derived from segments of

DNA discovered by chance within anony-mous (3)orgene-associated piecesof

geno-mic DNA (1, 4, 7). We report here that a

sequence present intheprotein IIIgene of the widely used M13 bacteriophage vector

(8) allows detection of a distinct set of

hypervariable minisatellites in human and animal DNA.

In the course ofa systematic search of RFLPs associated with the thyroglobulin

gene, we observed that a set of probes correspondingtoDNA segmentssubcloned in the 'bacteriophage M13 gave different resultswhen used in the classical hybridiza-tion mixture[Denhardt's medium

plus

her-ring sperm DNA (9)] or in a milk-based cocktail[Blottomedium(10)].A

complicat-ed andhighlypolymorphic patternwas

sys-B C

10 11 1 2 3

tematically observed with the enzyme Hae III in the lattercondition. It soon became evident thatthe pattern was unrelated to the nature of theinsert and thatwild-type M13 bacteriophages gave the same results. Nine unrelatedindividuals displayeddifferent pat-tems while monozygotic twins were indis-tinguishable (Fig. 1, A and B). Similar blots hybridized with an M13 probe in the pres-ence of herring sperm DNA showed no

polymorphism (Fig. 1C).

The logical explanation for this finding

wasthat a segment in M13hybridized to a

hypervariable minisatellite that could be competed forby fish DNA.If this were true,

G. Vassart, R. Monsieur, H. Brocas, D. Christophe,

InstitutdcRechercheInterdisciplinaire, Universite Libre

de Bruxelles, Campus Erasme, RoutedeLennik808, 1070 Bruxelles.

M.Georges andA. S. Lequarre, Departementde

G&nEti-que, Faculte de Medecine Vt&rinaire, Universite de

Lige,ruedesVeterinaires45, 1070Bruxelles.

A 1 2 3 0: 0000;; CEdDD; 4 5 6 7 8 5 6 B 4 5 6 4940 00 f00: 0 4334 3975 2895 1I*: 2289 1809 952 -20 -10 -5 -4 -3 -2 -1 kb

Fig. 1.Hypevariable polymorphic patternsdetected in human DNAwithwild-type M13 DN probe. Individuals 1to9areunrelatedCaucasians; 10 and 11aremonozygotic twins. Hae III-dige DNA(5,ug) from white blood cellswasseparated ina1% agarosegelin40 mMtris-acetate, 1 EDTA,pH 7.7,ataconstantvoltage of 9 V/cm for about 4 hours. The gelsweresoaked in 1.5MN

0.5M NaOH(2 x 30minutes) followed by equilibrationin IMNH4acetate,0.03MNaOH(2 minutes)and transferovernightontonitrocellulose filters(Schleicher &Schuell)in thesamesolui Single-stranded M13 (200 ng) was labeled with "2P-dATP to a specific activity of 0.5x

10-1.0x 10-9 cpm per microgram of DNA (13). After a 2-hour prehybridization, the filters X hybridized overnightat42°C in the following solutions: (A and B) 40%formamide, 6x SSC (stan4

salinecitrate), EDTA 5 mM, 0.25%dried skimmedmilk; (C)samemediumexceptthat thepowd

milkwasreplaced by 5x Denhardt'sanddenaturedherringspermDNA(100,ug/ml). The filters X allwashed under identical conditions: twice for 15 minutes in 2xSSC,0.1%SDS,roomtemperat four times for 15minutesin 2x SSC,0.1%SDS,65°C;and twice for 30minutesin1x SSC, 6

FilterswereexposedtoKodak XAR-5filmsat-70°C withanintensifyingscreen.

- 20

Fig. 2. IdentificationofM13sequences

hybridiz--10 ingtoherringspermDNA. M13RF DNAwas

5 digested with Xmn I (lanes 1 and 4), BstNI(lanes

4 2and5), and ClaI(lanes3 and6).Two identical

blots were prepared and hybridized to a total 3 M13probe (A)ortoM13sequencesselectedby

2 hybridization toherring spermDNA(B). M13

RFDNA(200 ng)wasdigestedwith restriction endonucleases and identical blotswere prepared asdescribed(Fig. 1). (A) The filterwas

hybrid--1 izedto atotal P-labeled M13

probe.

(B)

The kb filter was

hybridized

to labeled M13 sequences

that had beenselectedby hybridizationtoherring

sperm DNA. A 100-cm2 nitrocellulose filterwas

Aas saturated withherringspermDNAby soakingit

ested for 30 minutesinto NH4Ac lM-NaOH 0.03M mM containing 2.5 mg/mil denatured herringsperm

ZaCl, DNAand bakedat80°C for 2 hours. The filter

x30 wasprehybridizedfor2hours in 40%formamide,

tion. 6x SSC,5mMEDTA, 0.25%powdered milkat

9to 42°C, and hybridized overnight toa total

32P-were labeled M13 DNA probe. After washing, the

dard affinity-purified M13 sequences were eluted by lered boiling the filterfor 10 minutes in 10 ml of 10 were mMtris HCIpH 8, 1mMEDTA. About 0.1% of

ture; the inputradioactivitywasrecovered. Hybridiza-5°C. tion and washing offilterswas asdescribed inFig.

1A. 6 FEBRUARYI987 A 1 2 3 4 REPORTS 683 on May 3, 2009 www.sciencemag.org Downloaded from

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A 6862 61 B 1833 1 I GAGGGTGGTGGCTCT 6433x/ \ 2 \GAGGGTGGCGGTTCT 1013 3 GAGGGTGGCGGTTCT 4 GAGGGTGGCGGTaCT 5940

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1894

2283 5 GGcGGCGGCTCT 6 GGTGGTGGTTCT 7 GGTGGCGGCT'CT 8 GAGGGTGGTGGCTCT 1965 9 GAGGGTGGCGGTTCT 10 GAGGGTGGCGGCTCT 11 GAGGGaGGCGGTTCc 2246 12 GGTGGTGGCTCT 13 GGTtcCGGT 2-401

Cla I 265 Consensus AGGGTGGXGGXTCT

HaeIII 0 sequence I

(GluGlyGlyGlySer )n

Xmn v

Fig. 3. (A) M13 restriction map. The thick lines show the restriction fragments that werepreferentially

recognized bytheaffinity-purifiedM13sequences. The open boxesgive the localization of the tandem repeats.(B)Alignmentof tandem repeats present in the protein III gene ofM13. The coordinates are as described in (14).

14 15 16 17 18 19 B 1 2 3 4 5 6 7 8

t -

St.-~~w ....

Fig. 4. Hypervariable polymorphic patterns detected in human (A) and bovine (B) DNA by hybridizationto atandemrepeatpresentinM13 (15). (A) Individuals 12to 17are members ofa Caucasianpedigree,18and 19aremonozygotic twins; (B) 1and2aremonozygotictwinsobtainedby embryo transferofasurgicallybisected bovinemorula;bovines 3to8areunrelated animals from the

"BelgianWhiteandBlue"breed. The280-bpM13 HaeIII-Cla Irestrictionfragmentwasobtained

afteragarosegel electrophoresisandelectroelution.Thefragment(100 ng)waslabeled with32ptoa specific activity of 0.5 x 10- to1 x 109cpm permicrogramof DNA(13).Thisprobewasused in Southernblottingexperimentsasdescribed in the legendtoFig. 1 with themilk-based cocktail for

hybridization.

it should be possible to affinity-purify the bacteriophagesequenceinvolvedbyaround

ofhybridization-selection toherringsperm DNA. A radioactively labeled M13 probe

waspurifiedinthiswayandhybridizedtoa

Southern blot containing restriction

frag-mentsofthereplicative formofM13 (Fig.

2B). Total, unpurified M13 was used as a control on a replicate blot (Fig. 2A). The

results indicated that the affinity-purified 684

probe was enriched in sequences

corre-sponding to a region between coordinates

1013and2528ontheM13map(Fig. 3A).

Inspection ofthis region ofthe phage

se-quence revealed the presence of a 15-bp

motifrepeated in tandem at two places in theprotein III gene and corresponding to (Glu, Gly, Gly, Gly, Ser)n (Fig. 3B).A 280-bpfragment essentiallymade ofoneof the

repeat clusters was purified and used as a

probe on a Southern blot of humanDNA cleaved with Hae III. A typical

hyperpoly-morphic pattern was obtained (Fig. 4A). The pattern was clearly different from that obtained with Jeffreys' probe (5, 6) under

similarconditions. Highlypolymorphic

pat-terns were also obtained with DNAof bo-vine (Fig. 4B), equine, murine, and canine origin (11), whichextendstheusefulness of

the M13 to pedigree analyses in animal breeding.

Our results leadtothesurprising conclu-sion that one of the most commonly used

DNA vectors is able, by itself, to detect hypervariable minisatellites in a variety of mammals. The observation that fishDNA, which is frequently added in excess to

hy-bridization mixtures, competes for the

probe explains why this phenomenon had

remained unnoticed. It also suggests that

omission of carrier DNAduring

hybridiza-tion should help in finding additional

probes of this type. Finally, the wide conser-vation of sequences showing

cross-hybrid-ization withtheM13repeat posesthe ques-tionoftheir putativeroleormechanismof

maintenanceduringevolution. An interest-ing clue may befoundinthe recent discov-ery

that

anotherhighly conserved

minisatel-lite

isassociatedwithtransposableelements

(12).

REFERENCESAND NOTES

1. A. J. Jeffreys, V. Wilson, S. L. Thein, Nature

(London) 314,67(1985).

2. D. R.Higgs,S. E. Y.Goodbourn,J. S. Wainscoat,

J. B.Clegg, D.J.Weatherall, NuceicAcids Res. 9,

4213(1981).

3. A. R. Wyman and R.White,Proc.Natl. Acad.Sci.

U.SA.77, 6754(1980).

4. S. E.Y.Goodbourn,D. R.Higgs, J.B.Clegg,D.J.

Weatherall,ibid.80, 5022 (1983).

5. A.J. Jeffreys,J. F. Y. Brookfield, R. Semeonoff,

Nature(London)317,818(1985).

6. P.Gill,A.J.Jeffreys,D.J.Werrett,ibid.318,577

(1985).

7. G.I.Bell,M.J.Selby,W.J. Rutter,ibid.295,31

(1982).

8. J.Messing, B.Gronenborn,B.Muller-Hill, P. H.

Hofschneider,Proc.Natl.Acad. Sci.U.SA.74,3642

(1977).

9. T. Maniatis,E. F.Fritsch, J. Sambrook,Molecular

Clonitg:ALaboratoryManual(ColdSpnrngHarbor Laboratory, Cold Spring Harbor, NY,1982).

10. D. A.Johnson,J. W. Gautsch, J.R.Sportsman, J.

H.Elder, GencAnal.Technol.1,3(1984).

11. M.Georgesetal.,inpreparation.

12. D. Liebermann, B. Hoffman-Liebermann, A. B.

Troutt,L. Kedes,S.N.Cohen, Mol. Cell. Biol.6,

218(1986).

13. A. P.Feinberg and B. Vogelstein, Anal.Biocaem.

132,6(1983).

14. P. M, vanWezembeck, T.J.M.Hulsebos, J. G.

Schoenmakers,Gene11,129(1980).

15.Thesequenceandproceduredescribed inthe

pre-sentstudyarethesubjectofapendingpatent. 16.Thecontnuoussupportand cnticalinterestofJ.E.

Dumontand R.Hansetaredeeplyacknowledged.

We thank A. Deplano and M. Mairie for expert

technical assistance and D. Leemansfor the

prepara-tionof themanuscript.WearegratefultoA.Massip forproviding thebovine monozgtictwins. Sup-portedbyActionsConcert6es(Minist&edela

Poli-tique Scientifique) and AssociationRecherche

Bio-medicaleetDiagnostica.s.b.l.

29September1986;accepted12December1986

SCIENCE,VOL. 235 A12 13

on May 3, 2009

www.sciencemag.org

Figure

Fig. 1. Hypevariable polymorphic patterns detected in human DNA with wild-type M13 DN probe
Fig. 4. Hypervariable polymorphic patterns detected in human (A) and bovine (B) DNA by hybridization to a tandem repeat present in M13 (15)

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