Review article

Journal of Medical Genetics, 1984, 21, 164-172

Blot hybridisation analysis of genomic DNASANTIE VANDENPLAS, IAN WIID, ANNE GROBLER-RABIE,KIRSTY BREBNER, MICHAEL RICKETTS, GILLIAN WALLIS,ANDRE BESTER, CHARLES BOYD, AND CHRISTOPHER MATHEWFrom the MRC Unit for Molecular and Cellular Cardiology, University of StellenboschMedical School, PO Box 63, Tygerberg 7505, South Africa.

SUMMARY Restriction endonuclease analysis of specific gene sequences is proving to be a valuabletechnique for characterisation and diagnosis of inherited disorders. This paper describes detailedprotocols for isolation, restriction, and blot hybridisation of genomic DNA. Problems andalternatives in the procedure are discussed and a troubleshooting guide has been provided to helprectify faults.

The development of techniques for the cloning1 andanalysis2 of genes from complex organisms laid thefoundation for the study of mutant genes associatedwith human inherited disorders. DNA from a personcan now be cleaved into fragments of defined lengthby restriction endonucleases. The fragments arethen separated by gel electrophoresis, blotted ontofilters,2 and incubated with radioactively labelledgene specific probes. These probes, obtained bymolecular cloning techniques, are isolated andcharacterised DNA sequences which will associatespecifically with homologous genomic DNA se-quences on the filter. Thus, only fragments con-taining part or all of the gene of interest will bedetected. This new recombinant DNA technologywas rapidly applied to the molecular characterisa-tion and antenatal diagnosis of the haemoglobino-pathies and thalassaemia.3-7 As cloning techniqueshave become more sophisticated, the number ofpurified, cloned human genes has proliferated tothe extent that a recently published list8 is alreadyout of date. A considerable number of geneticdiseases are therefore amenable to DNA analysis,and the use of linked restriction fragment lengthpolymorphisms9 10 has further extended the applic-ability of the technique.

These advances have brought DNA analysiswithin the scope of the clinical geneticist, and thetechniques will ultimately become part of theroutine service provided by human genetics depart-ments. DNA blotting and molecular hybridisationReceived for publication 25 September 1983.Accepted for publication 12 October 1983.

do, however, present a considerable technicalchallenge to workers new to the field. Furthermore,although standard protocols have been described,1 12they do not provide details of all aspects of genomicDNA analysis or a troubleshooting guide for thenon-specialist.

In this paper, we present details of a protocolwhich works reproducibly in our hands and discusssome of the difficulties and alternatives with whichthe researcher may be confronted.

MaterialsCHEMICALS AND EQUIPMENTChemicalsRestriction enzymes were obtained from BethesdaResearch Laboratories, Boehringer-Mannheim, andNew England Biolabs. Bovine serum albumin,fraction V, and pancreatic ribonuclease A werepurchased from Bayer-Miles. Sigma providedherring sperm DNA, polyadenylic acid, polyvinyl-pyrrolidone (type 360), and ficoll (type 400).Nitrocellulose (BA 85, 0 45 ,m) was obtained fromSchleicher and Schuell. Nuclease free bovine serumalbumin, proteinase K, T4 DNA polymerase,crystalline redistilled phenol, X DNA, and the nicktranslation kits were purchased from BethesdaResearch Laboratories. Agarose (Seakem) wasobtained from Marine Colloids. [a32P]-deoxycytidinetriphosphate (3000 Ci/mmol, 10 mCi/ml in astabilised aqueous solution) was purchased fromAmersham.

All chemicals not further described were ofAnalar (or equivalent) grade.

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EquipmentA horizontal gel electrophoresis apparatus based onthe design described by Southern2 was used. Thedimensions of the gel mould were 183 mm (width)x 170 mm (length). The teeth of the 'comb' or wellformer were 10 mm in width and 1 mm thick. Thegel chamber and 'comb' were constructed of Perspexwith platinum wire electrodes.The hybridisation chamber was made of Perspex

and designed by Alec Jeffreys (Leicester University)(see fig 1 for details).Kodak X-Omatic x-ray cassettes with Fuji Macl 2

calcium tungstate intensifying screens were used.The films used were 'Cronex Safety' and KodakX-Omat AR.The transilluminator (model C62) was obtained

from Ultra-violet Products Inc, San Gabriel,California.

SOLUTIONS

(1) Cell lysis buffer320 mmol/l sucrose.

A

I 0

ring

pointlid

100n+160mm

B

0]6 mm]4mm

ring

24mm

]5mm

40mm

100 mm

FIG 1 Diagram of the hybridisation chamber designedby Alec Jeifreys showing the plane (A) and elevation (B).

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1 % (v/v) triton X-100.5 mmol/l MgC12.10 mmol/l Tris- HCI, pH 7 6.

(2) Saline-EDTA (pH 8 0)25 mmol/l EDTA.75 mmol/l NaCI.

(3) Phenol:chloroformTo a phenol:chloroform (1:1 v/v) mixture add 0 5volume 1 x TE (solution 16). Store at 40C in alight-tight bottle.

(4) 10 x gel electrophoresis buffer0-89 mol/l Tris-borate.0i89 mol/l boric acid.0-02 mol/l EDTA.(108 g Tris base, 55 g boric acid, and 40 ml 0 5 mol/lEDTA, pH 8 - 0, per litre H20).

(5) Ficoll-Orange G0a1 % (w/v) Orange Gin 20% (w/v) ficoll.10 mmol/lEDTA (pH 7 0).

(6) Chloroform:octanol(24:1 v/v).

(7) Restriction endonuclease bufferPrepare as a 10 x stock according to manufacturer'sinstructions.

(8) Denaturation solution0 5 mol/l NaOH.I1 5 mol/l NaCI.

(9) Neutralisation buffer0 5 mol/l Tris-HCI, pH 55.3.0 mol/l NaCI.0 3 mol/l sodium citrate.

(10) 20 x SSC3 0 mol/l NaCl.0 3 mol/l sodium citrate, pH 7 0.

(11) 100 x Denhardt's2% (w/v) bovine serum albumin fraction V.2% (w/v) polyvinylpyrrolidone type 360.2% (w/v) Ficoll type 400.Heat to 400C while stirring, followed by gentlestirring at 40C overnight.

(12) Hybridisation solution3 x SSC.10 x Denhardt's.0a 1% SDS (sodium dodecyl sulphate).10 ,ug/ml polyadenylic acid.

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50 ,ug/ml heat denatured sonicated herring sperm.

DNA (see solution 14).

(13) Post-hybridisation wash solution3 x SSC.10 x Denhardt's.0-1 % sodium dodecyl sulphate.

(14) Heat denatured sonicated herring sperm DNA2 mg/ml stock solution.Dissolve and sonicate to an average length ofapproximately 600 base pairs. (Determine by agarose

gel electrophoresis. See Method section on agarose

gel electrophoresis of DNA restriction fragments.)Denature in boiling waterbath for 10 minutes. Coolrapidly on ice.

(15) Stringent wash solution0-1 % (w/v) sodium dodecyl sulphate.0 1 x SSC (see discussion).

(16) 1 x TE10 mm Tris-HCI, pH 7-5.1 mmol/l EDTA.

(17) 10 x T4 polymerase buffer330 mmol/l Tris-acetate, pH 7 9.660 mmol/l potassium acetate.100 mmol/l magnesium acetate.1 mg/ml nuclease free bovine serum albumin.5 mmol/l dithiothreitol.The following stock solutions are also recommended:

(a) 10% (w/v) sodium dodecyl sulphate.(b) 10 mg/ml proteinase K.(c) 10 mg/ml ribonuclease, heat treated at 800C for10 minutes.(d) 10 mg/ml ethidium bromide.(e) 0 1 mol/l EDTA, pH 7 0. Adjust pH in order to

dissolve EDTA.(f) 5 mol/l sodium perchlorate.

Methods

PREPARATION OF GENOMIC DNAHuman DNA was isolated from lymphocytes usinga procedure modified from Kunkel et al.13A total of 10 ml of whole blood was collected in

vacutainer tubes containing EDTA or citrate as

anticoagulant and added to 60 ml of lysis buffer.This suspension was then gently homogenised in a

Dounce homogeniser (5 strokes up and down). Thenuclei were pelleted by centrifugation at 2500 g for20 minutes at 40C. The nuclear pellet was suspendedin 8 ml of 25 mmol/l EDTA, 75 mmol/l NaCI, pH8-0, using a sterile pipette. After the addition of800 ,ul of 10% (w/v) sodium dodecyl sulphate and0-I ml of the 10 mg/ml proteinase K solution, themixture was incubated for 2 hours at 370C. A total

Santie Vandenplas et al

of 500 V1 of a 5 mol/l sodium perchlorate solutionwas added. The digest was gently mixed with 8 mlof phenol :chloroform until homogeneous. Thephases were separated by centrifugation for 10minutes at 12 000 g at 100C. The upper, aqueous,phase was removed and further extracted with anequal volume of chloroform:octanol (24:1). Thephases were again separated after gentle mixing.DNA was precipitated from the aqueous phase byadding 2 volumes of cold absolute ethanol. Theprecipitate was lifted out with the sealed end of aPasteur pipette and shaken into 1 x TE. The DNAwas allowed to dissolve overnight at 40C. A total of0o 1 volumes 20 x SSC and 0 01 volumes 5 mg/mlribonuclease were added and the mixture incubatedfor 1 hour at 370C. Then, 2 ml of sterile water wasadded and the solution was extracted twice withchloroform:octanol (24:1). The DNA was pre-cipitated by adding 2 volumes of absolute ethanoland washed twice with 70% ethanol. The DNApellet was dried under a vacuum for 15 minutes andfinally dissolved in 0 5 ml of sterile double distilledwater.The DNA concentration is estimated by the

determination of its absorbance at 260 nm assumingthat its Alm, 260 is 200 (that is, a 1 g/100 mlsolution in a 1 cm lightpath has an absorbance at260 nrm of 200).14PREPARATION OF PROBE DNADetails of the isolation of recombinant plasmidscontaining specific sequence probes are given byManiatis et al.12 Approval for these experiments bythe Genetic Manipulation Advisory Group (GMAG)is required.

RESTRICTION ENDONUCLEASE DIGESTIONOF GENOMIC DNAAn incubation mixture with a final volume of 50 Vl1was prepared containing 10 ,g DNA, 0 1 volume of10 x restriction endonuclease buffer, 30 unitsrestriction enzymes (3 U/Vg DNA), and 100 ,ug/mlnuclease-free bovine serum albumin. This was thenincubated for 16 hours at the temperature requiredfor the enzyme used. The condensate was collectedby centrifugation for 10 seconds in an Eppendorfmicrocentrifuge. Digestion was terminated by theaddition of 0*1 volumes of a 100 mmol/l EDTAstock (pH 7 0).A 5 p1 aliquot was removed from each incubation

mixture, mixed with 1 V.l of Orange G-ficoll, andelectrophoresed in order to determine whetherdigestion was complete (see Discussion).AGAROSE GEL ELECTROPHORESIS OF DNARESTRICTION FRAGMENTSFirstly, 0 6% agarose horizontal slab gels were

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prepared by adding 1 2 g of agarose powder to200 ml of 1 x electrophoresis buffer and boilinguntil completely dissolved. The solution was thencooled to 650C and ethidium bromide added to aconcentration of 1 ,ug/ml. The molten agarose waspoured into the gel mould with comb in place (0 5mm off gel bed) and allowed to set at room tempera-ture for 1 hour.A total of 5 ,ul ficoll-Orange G solution was

added to each DNA digest. The samples were thenapplied to the gel and electrophoresed (in 1 xelectrophoresis buffer, containing 1 ,ug/ml ethidiumbromide) at 30 V for 30 minutes at constant voltage.After the samples had entered the gel, it was sub-merged in electrophoresis buffer and the electro-phoresis continued overnight. About 10 000 cpm of aradiolabelled DNA molecular weight marker (seeT4 DNA labelling, Methods) was applied to onelane of the gel before electrophoresis.

After electrophoresis was completed (Orange Ghad migrated to end of gel), the gel was placed on aUV transilluminator and photographed, using anorange filter.

TRANSFER OF DNA FROM AGAROSE GELONTO NITROCELLULOSEThe original method as described by Southern2 wasused with a few modifications.

Pre-treatment ofgelThe DNA in the gel was denatured by submersionin 300 ml denaturation buffer for 2 hours with gentleshaking. The gel was briefly rinsed with distilled

..weights..

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167

water and then neutralised by submerging in 300 mlneutralising buffer for 1 hour with gentle shaking.

DNA transferDetails of the DNA transfer system are shown infig 2.A tray was filled with 20 x SSC and a glass plate

was supported in the tray. A piece of Whatman 3Mfilter paper was draped over the glass plate with thesides in contact with the SSC solution. The top ofthe filter paper was also soaked with 20 x SSC. Apiece of nitrocellulose was then cut to the size ofthe gel using a sterile blade. (Nitrocellulose must behandled with forceps or washed gloves.) The nitro-cellulose was wet by flotation on 2 x SSC solutionfor 5 minutes. The pre-treated gel was then carefullyslid onto the wet filter paper on the glass plate,taking care to avoid trapping air beneath it. Thepaper around the gel was then covered with a layerof waterproof film (for example, Saran wrap, clingfilm, etc). Excess liquid was removed from the gelsurface and the soaked nitrocellulose was placed onthe gel, taking care not to trap air beneath it.Another two pieces of Whatman 3M paper were

cut according to gel size and soaked in 2 x SSC.These were then placed on top of the nitrocellulose(making sure no air was trapped beneath). Dryabsorbant paper was placed on top of the filterpaper and compressed with weights (i 1 kg).The time allowed for transfer was about 40 hours

at 40C. During this time the level of SSC in the traywas checked and the wet absorbant paper replacedby dry paper.

i glass plate

FIG 2 DNA transfer system.Strips of Saran wrap aresuspendedfrom the outer edges of

br the gel to the sides of the tray sothat SSC is forced to movethrough the gel, and to prevent

wrap evaporation of the SSC.

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After transfer was completed, the position andorientation of the lanes of DNA in the gel were

marked on the cellulose nitrate sheet with a blackwaterproof marker pen. The sheet was then cut intorectangular strips (two lanes per strip) to fit intothe hybridisation chamber. The filters were thensoaked in 2 x SSC for 10 minutes and baked at800C for 2 hours. (The filters should not be allowedto adhere to each other during soaking.)

HYBRIDISATIONThe protocol described by Jeffreys and Flavell15 wasused for washing and hybridisation of the filters.

Pre-hybridisation washesThe baked nitrocellulose strips were wet by flotationon a 3 x SSC solution at room temperature. Thefilters were then incubated with gentle shaking at650C for 30 minutes in 50 ml preheated 3 x SSC.The filters were then washed in 50 ml of a solutioncontaining 3 x SSC, 10 x Denhardt's (preheated to650C) for 60 minutes. Finally, the filters were

washed for 30 minutes at 650C in 50 ml of pre-heated hybridisation solution.

HybridisationHybridisation was carried out in a hybridisationchamber as described in the Materials section. Atotal of 10 ml of the hybridisation buffer was placedin the chamber together with the heat denaturedradioactively labelled probe (see next section). Thewashed filters were carefully placed into this mixtureand the unit sealed and incubated at 650C withgentle shaking for 40 hours.

RADIOLABELLING OF DNA

Nick translation of the DNA probeNick translation16 was performed with 100 pCi of[a-32P]dCTP (specific activity of 3000 Ci/mmol,concentration 10 mCi/ml). The standard nicktranslation labelling protocol, supplied with the kitobtained from Bethesda Research Laboratories,was followed.A total of 5 ,ul of a solution containing 0 - 2 mmol/l

dNTPs (dATP, dTTP, dGTP) was pipetted into a

1.5 ml microcentrifuge tube on ice. Then, 0 5 ,gofprobeDNA and 100 ,uCi of radioactive nucleotidewas added, the mixture was made up to a volume of45 pl with sterile distilled water, and 5 0d of amixture of the DNA polymerase I (0 4 U/,u) andDNAse I (40 pg/[tl) was added. The solution wasmixed gently but thoroughly. It was centrifugedbriefly (microfuge 15000 g for 5 seconds) and in-cubated at 150C for 60 minutes. The reaction was

stopped by the addition of 5 ,l Stop Buffer (300mmol/l Na2EDTA, pH 8 0) and extracted with an

Santie Vandenplas et al

equal volume of phenol:chloroform. The phenolphase was re-extracted with 100 V. 1 x TE. Thelabelled DNA in the pooled aqueous phases wasthen separated from the free nucleotides by chroma-tography on a small column of Sephadex G-50(medium), poured in a Pasteur pipette, and equili-brated with 3 x SSC. The DNA was eluted with3 x SSC. Twenty fractions (three drops) werecollected and 1 0 Vul of each fraction was counted.The excluded peak containing the labelled DNA waspooled, an aliquot was counted, and the specificactivity of the probe calculated.

T4 DNA polymerase labelling of?DNAXDNA was labelled with [c- 32P] dCTP for use as aDNA molecular weight marker. A total of 6 ,ug of?DNA was digested with 18 units of Hind III for90 minutes at 370C in an incubation volume of 60 ,dwhich contained 6 [±l 10 x T4 polymerase buffer.After incubation, 20 [,l was run on a 0 6% agarosegel to determine if digestion was complete.

Then, 2 ,ug of the digested XDNA (20 ,d of theHind III digest) was added to 2 5 units of T4-DNApolymerase and incubated for 5 minutes at 370C.After this, 1 ,tl of a solution containing 2 mmol/ldGTP, 2 mmol/l dATP, 2 mmol/l dTTP, and I ,uCi[oc-32P] dCTP was added and incubated for 1minute at 37°C. Then, 5 ,d cold 1 mm dCTP wasadded and incubated for 10 minutes at 37°C. Thereaction was terminated by incubating for 5 minutesat 70°C. This mixture was then phenol extracted andthe DNA isolated on a Sephadex G-50 (medium)column as described in the previous section.A total of 1 x 104 cpm of 32P-labelled Hind III

digested XDNA was used as a DNA molecularweight marker (see fig 3).

POST-HYBRIDISATION WASHESAfter hybridisation, the filters were washed inpost-hybridisation buffer; 300 ml of post-hybridisa-tion buffer preheated to 650C was divided equallyamong six containers. The filters were then washedfor 1 minute at 650C in each of four containers, thenwashed for 30 minutes each in the remaining twocontainers. The filters were finally given 2 x 30minutes stringency washes at 650C in 50 ml ofstringency buffer. The wet filters were then alignedand heat sealed within a plastic bag.The filters and x-ray film were placed in a cassette

between two intensifying screens and the film wasexposed at - 700C for 1 to 14 days.

RE-USE OF FILTERSUsed filters can be rehybridised to a second probeafter removal of the original probe with NaOH.Filters were soaked in denaturation solution

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23 720

946C0-5

6670-

4260-

virus.18 Transformed cells can be stored in liquidnitrogen, thus providing a constantly renewablesource of DNA.The yield of DNA obtained will depend on the

white cell count of the patient, but is generally150 to 500 F±g per 10 ml of whole blood. An ab-sorbance scan of the DNA (220 to 300 nm) shouldbe run to determine whether impurities such asphenol or proteins are present (see fig 4). DNAsamples contaminated with phenol should be re-extracted with CHCl3. Protein can be removed byrepeating the phenol and chloroform extractions.The DNA solution can be stored at - 700C or-200C.

2250 - - .960 -

59C-RESTRICTION ENDONUCLEASE DIGESTIONIt is important to obtain complete digestion of theDNA by the restriction enzyme. Partial digests will

i2 3 4 5 6 produce spurious high molecular weight DNA123 4 5 6 fragments. The completeness of digestion should be

checked by electrophoresis of an aliquot of theFIG 3 Detection of specific genomic sequences by digest. Examples of complete and partial digests areblot hybridisation. DNA was digested with restriction shownti fig

ofPartial digests aieenzymes, electrophoresed on 0-6% agarose gel, blotted, shownzn fig 5. Partial dgests can also be obtainedand hybridised with a pro o2 (1) collagen cDNA probe.7 if enzyme action is affected by methylation of theLane 1. 32P-labelled X molecular weight marker. DNA (fig 5). In this cage an enzyme which recognisesLanes 2-6. ProC2 (1) collagen sequences. (This result the same sequence, but which is not affected byw'as obtainedfrom an overnight exposure.) methylation, should be used.

(solution 8) for 5 minutes, neutralisation buffer(solution 9) for 2 hours, and finally 3 x SSC for15 minutes. They were then baked at 800C for 2hours before being prehybridised in the usual way.

Discussion

We will discuss each step in the procedure withregard to possible problems and alternatives.

CHEMICALSIt is probable that chemicals from suppliers otherthan those mentioned can be used. However, thisshould be established in controlled experimentsbefore a chemical from a different supplier is usedroutinely.

PREPARATION OF GENOMIC DNAWhole blood is a convenient source of DNA. If theblood is collected in EDTA, which inactivatesendogenous nucleases, it can be stored at 40C for atleast 7 days before isolation of the DNA. Whiteblood cells prepared from buffy coats or ficollgradients can be stored in physiological saline at-700C for several years.Permanent cell lines of patients can be obtained

by transformation of lymphocytes with Epstein-Barr

AGAROSE GEL ELECTROPHORESISA variety of designs of apparatus are suitable forelectrophoresis of the DNA. Horizontal slab gelsare convenient for gels of low agarose concentration.A number of such apparatuses are commerciallyavailable (for example, BRL, Bio-Rad) but they caneasily be constructed by hospital or university

0 6

0 5C,

41 0*4

D03

02

A

280 260 280 260Wavelength (nm)

C

280 260

FIG 4 Absorbance spectrum ofhuman DNA.A. Spectrum of uncontaminated DNA showingabsorbance maximum at 260 nm. B. Phenolcontaminated DNA showing broadening of the peak asa result of absorption by phenol at 270 nm. C. Proteincontaminated DNA showing protein absorbanceshoulder at 280 nni.

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migrates faster and causes less quenching of theethidium bromide fluorescence.1"

1 2 2

FIG 5 Restriction endonuclease digestion ofhumangenomic DNA. A. Ethidium bromide staining ofEcoRl digests. Lane I shows discrete bands ofhighlyrepeated DNA sequences indicative of completedigestion. Lane 2 shows DNA partially digested byEcoRI. B. Hybridisation of the EcoRI digested DNAin A with a collagen pro O2 (1) cDNA probe.17C. Partial digestion ofgenomic DNA as a resiult ofmethylation. Lane 1. DNA digested with Msp I showingcomplete digestion and the correct fragment pattern.Lane 2. DNA from the same person digested withHpa II which recognises the same base sequence asA-Isp 1, but will not cut if the internal cytosine of thissequence is methylated.

workshops. Gels should be at least 150 mm inlength to ensure accurate measurements of DNAfragment sizes and high resolution of the bands.Resolution will also be improved by electrophoresisat a low voltage for 12 to 16 hours rather than at ahigh voltage for a shorter period. Changes of pHwhich occur as a result of electrophoresis can beminimised by using a gel tank which holds a largervolume of buffer (2 to 3 1). Alternatively, the buffercan be circulated through the cathode and anodecompartments.

Glycerol or sucrose are often added to therestriction digests before loading on the gel toincrease the density of the solution. However, theselow molecular weight solutes cause streaming ofthe sample up the side of the well, which leads tothe production of U shaped DNA bands. Ficollavoids this effect. Bromophenol blue or Orange Gcan be used as a sample marker dye, but Orange G

TRANSFER OF DNA ONTO NITROCELLULOSEThe rate of transfer of DNA out of the gel dependson DNA size and the thickness and agarose con-centration of the gel. Large fragments (>10 kb) aretransferred very slowly. They may be broken downin the gel before transfer either by irradiation of theDNA on the transilluminator for 5 to 10 minutes orby partial depurination with dilute acid followed bystrand cleavage with alkali.19 The partial depurina-tion procedure is not usually necessary, but may beincluded if large restriction fragments are to bedetected. If fragments of < 10 kb are to be detected,an overnight transfer (without depurination) issufficient.The type of nitrocellulose paper used can signifi-

cantly influence the sensitivity of detection of DNAfragments. We have found that Schleicher andSchuell nitrocellulose binds DNA more efficientlythan several other brands.Once the filters have been baked after transfer,

they can be stored for several months at 40C beforehybridisation with the DNA probe.

HYBRIDISATION OF PROBE TO FILTERSIlybridisation can be carried out in a perspex box(fig 1) or a sealed plastic bag. We have found thatthe slight inconvenience of cutting up the filters tofit the hybridisation box is more than adequatelycompensated by the lack of background signalobtained. The filters should be well covered by theprobe solution during hybridisation and should notbe allowed to dry out until after the final stringentwashes have been done.The time required for adequate hybridisation

depends on the concentration and sequence com-plexity ofthe probe, the temperature ofhybridisation,and the salt concentration of the probe solution.'220An overnight hybridisation is sufficient to detectsingle copy genomic sequences using the conditionsdescribed here (see methods), provided that thespecific activity of the probe is not less than 1 x 108cpm/,Vg DNA. However, we routinely hybridise forabout 40 hours.

Sequence specific probes are generally recom-binant plasmids containing complementary DNA(cDNA) or genomic DNA sequences. Before usingsuch a probe for the first time it is important toestablish that it contains the desired insert. This canbe done by comparing its electrophoretic mobilitywith that of the parental plasmid and checking thatit produces the expected fragment sizes afterdigestion with one or two restriction enzymes.Either the entire recombinant plasmid can be

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TABLE Troubleshooting guide.

Problem Possible causes Remedy

Atypical DNA scan Phenol or protein contamination Re-extract DNA with CHC13 or phenol and CHCI3DNA appears degraded on gel Blood kept too long before DNA extracted Check undigested DNA for degradation

Nuclease contamination of DNA, buffers, If degraded, obtain fresh bloodor enzyme If not, use fresh buffer and enzyme

DNA not digested Faulty buffer or restriction enzyme Check enzyme and buffer with ?DNAImpurities in DNA Re-extract DNA

Incomplete DNA transfer No Saran wrap between filter paper wick Repeat transferand nitrocellulose Increase transfer time

Air trapped between gel and filter Pre-treat gel with HCIHigh background signal in DNA lanes Post-hybridisation wash stringency too low Increase stringency of final washes

Probe contains repeat sequences Change probeHigh background all over filters Inadequate pre-hybridisation Repeat stringency washes

Handling filters without gloves If inadequate, remove probe with NaOH andDrying out of filters in contact with probe rehybridise

No bands detected on autoradiograph DNA not binding to nitrocellulose Include 32P-labelled ?DNA on gelIf not detected, change nitrocellulose and make

fresh 20 x SSCNo insert in plasmid probe Electrophorese 20-100 pg of probe on one

lane of gelIf signal obtained, prepare and characterisenew probe

Inadequate sensitivity of detection Remake solutionsIncorrect hybridisation or wash solutions Repeat nick translation with fresh 32p dNTP orSpecific activity of probe too low fresh enzymeProbe too short after nick translation Reduce DNAse concentration in nick translationProbe not denatured before hybridisation Denature probe

labelled, or the insert cut out and purified bypreparative gel electrophoresis. The latter approachhas the advantage that spurious restriction frag-ments resulting from contamination of humanDNA samples with plasmid DNA would not bedetected. If a particular probe consistently produceshigh background signals, a new stock of the re-combinant plasmid should be prepared from afresh culture.

After hybridisation the filters should be givenhigh stringency (low salt concentration) washes inorder to remove non-specifically bound probe andto reduce hybridisation to other related genesequences.20 We use 0-1 x SSC washes whenhybridising with a probe containing genomic DNAand 0 1 to 0-25 x SSC for cDNA probes.When a new probe is being used, a series of

washes can be carried out to establish a suitablestringency.

A UTORADIOGRAPHYThe conditions for autoradiography and the types ofx-ray film and intensifying screens available havebeen reviewed.21 We have found Kodak X-OmatAR, Fuji RX, and Cronex Safety films to be suitable,used with Dupont Cronex Lightning Plus or FujiMach 2 calcium tungstate intensifying screens. Notethat exposure at - 700C is more efficient than at-20°C.The length of exposure required to detect specific

sequences will depend on factors such as the specificactivity of the probe and the amount of uniquesequence DNA on the filter. Using the conditions

described here, faint bands can be detected afteran overnight exposure, whereas intense bands canbe seen after exposures of 3 to 7 days.

Conclusion

If the protocol described in this review is followedcarefully, results such as that illustrated in fig 3should be obtained routinely. However, problems dooccur, especially when first setting up this technique.A troubleshooting guide is given in the table toassist in diagnosing and rectifying the problem.

This work was supported by grants from the SouthAfrican Medical Research Council. The cDNAclone for human proa2 (1) collagen was supplied byDrs F Ramirez and D Prockop of Rutgers MedicalSchool, New Jersey. We would like to thank MrsR van Dyk for her assistance with the preparationof the manuscript.

ReferencesCohen SN, Chang ACU, Boyer HW, Helling RB. Con-struction of biologically functional bacterial plasmids invitro. Proc NatlAcad Sci USA 1973 ;70:3240-4.

2 Southern EM. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J MolBiol 1975 ;98 :503-18.Mears JG, Ramirez F, Leibowitz D, et al. Changes inrestricted human cellular DNA fragments containingglobin gene sequences in thalassaemia and related dis-orders. Proc NatlAcadSci USA 1978;75:1222-6.Orkin SH, Alter BP, Altay C, et al. Application ofendonuclease mapping to the analysis and prenataldiagnosis of thalassemias caused by globin gene de-letion. N EnglJMed 1978 ;299 :166-72.

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Correspondence and requests for reprints to DrC Mathew, MRC Unit for Molecular and CellularCardiology, University of Stellenbosch MedicalSchool, PO Box 63, Tygerberg 7505, South Africa.

Correction

In the Review article 'The fragile X syndrome: thepatients and their chromosomes' by De Arce andKearns published in the April 1984 issue of thejournal (vol 21, pp 84-91), the sentence starting11 lines from the bottom of p 87, column 2, shouldread: "From the work of Sutherland42 andothers,58 6162 we know that TC 199 induces theexpression of certain fragile sites and non-specificgaps on C group autosomes in normal controls".

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