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Blot Hybridisation Analysis of Genomic DNA

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Blot Hybridisation Analysis of Genomic DNA J Med Genet: first published as 10.1136/jmg.21.3.164 on 1 June 1984. Downloaded from Review article Journal of Medical Genetics, 1984, 21, 164-172 Blot hybridisation analysis of genomic DNA SANTIE VANDENPLAS, IAN WIID, ANNE GROBLER-RABIE, KIRSTY BREBNER, MICHAEL RICKETTS, GILLIAN WALLIS, ANDRE BESTER, CHARLES BOYD, AND CHRISTOPHER MATHEW From the MRC Unit for Molecular and Cellular Cardiology, University of Stellenbosch Medical School, PO Box 63, Tygerberg 7505, South Africa. SUMMARY Restriction endonuclease analysis of specific gene sequences is proving to be a valuable technique for characterisation and diagnosis of inherited disorders. This paper describes detailed protocols for isolation, restriction, and blot hybridisation of genomic DNA. Problems and alternatives in the procedure are discussed and a troubleshooting guide has been provided to help rectify faults. The development of techniques for the cloning1 and do, however, present a considerable technical analysis2 of genes from complex organisms laid the challenge to workers new to the field. Furthermore,copyright. foundation for the study of mutant genes associated although standard protocols have been described,1 12 with human inherited disorders. DNA from a person they do not provide details of all aspects of genomic can now be cleaved into fragments of defined length DNA analysis or a troubleshooting guide for the by restriction endonucleases. The fragments are non-specialist. then separated by gel electrophoresis, blotted onto In this paper, we present details of a protocol filters,2 and incubated with radioactively labelled which works reproducibly in our hands and discuss gene specific probes. These probes, obtained by some of the difficulties and alternatives with which molecular cloning techniques, are isolated and the researcher may be confronted. http://jmg.bmj.com/ characterised DNA sequences which will associate specifically with homologous genomic DNA se- Materials quences on the filter. Thus, only fragments con- CHEMICALS AND EQUIPMENT taining part or all of the gene of interest will be Chemicals detected. This new recombinant DNA technology Restriction enzymes were obtained from Bethesda was rapidly applied to the molecular characterisa- Research Laboratories, Boehringer-Mannheim, and tion and antenatal diagnosis of the haemoglobino- New England Biolabs. Bovine serum albumin, pathies and thalassaemia.3-7 As cloning techniques fraction V, and pancreatic ribonuclease A were on October 1, 2021 by guest. Protected have become more sophisticated, the number of purchased from Bayer-Miles. Sigma provided purified, cloned human genes has proliferated to herring sperm DNA, polyadenylic acid, polyvinyl- the extent that a recently published list8 is already pyrrolidone (type 360), and ficoll (type 400). out of date. A considerable number of genetic Nitrocellulose (BA 85, 0 45 ,m) was obtained from diseases are therefore amenable to DNA analysis, Schleicher and Schuell. Nuclease free bovine serum and the use of linked restriction fragment length albumin, proteinase K, T4 DNA polymerase, polymorphisms9 10 has further extended the applic- crystalline redistilled phenol, X DNA, and the nick ability of the technique. translation kits were purchased from Bethesda These advances have brought DNA analysis Research Laboratories. Agarose (Seakem) was within the scope of the clinical geneticist, and the obtained from Marine Colloids. [a32P]-deoxycytidine techniques will ultimately become part of the triphosphate (3000 Ci/mmol, 10 mCi/ml in a routine service provided by human genetics depart- stabilised aqueous solution) was purchased from ments. DNA blotting and molecular hybridisation Amersham. Received for publication 25 September 1983. All chemicals not further described were of Accepted for publication 12 October 1983. Analar (or equivalent) grade. 164 J Med Genet: first published as 10.1136/jmg.21.3.164 on 1 June 1984. Downloaded from Blot hybridisation analysis ofgenomic DNA 165 Equipment 1 % (v/v) triton X-100. A horizontal gel electrophoresis apparatus based on 5 mmol/l MgC12. the design described by Southern2 was used. The 10 mmol/l Tris- HCI, pH 7 6. dimensions of the gel mould were 183 mm (width) x 170 mm (length). The teeth of the 'comb' or well (2) Saline-EDTA (pH 8 0) former were 10 mm in width and 1 mm thick. The 25 mmol/l EDTA. gel chamber and 'comb' were constructed of Perspex 75 mmol/l NaCI. with platinum wire electrodes. The hybridisation chamber was made of Perspex (3) Phenol:chloroform and designed by Alec Jeffreys (Leicester University) To a phenol:chloroform (1:1 v/v) mixture add 0 5 (see fig 1 for details). volume 1 x TE (solution 16). Store at 40C in a Kodak X-Omatic x-ray cassettes with Fuji Macl 2 light-tight bottle. calcium tungstate intensifying screens were used. The films used were 'Cronex Safety' and Kodak (4) 10 x gel electrophoresis buffer X-Omat AR. 0-89 mol/l Tris-borate. The transilluminator (model C62) was obtained 0i89 mol/l boric acid. from Ultra-violet Products Inc, San Gabriel, 0-02 mol/l EDTA. California. (108 g Tris base, 55 g boric acid, and 40 ml 0 5 mol/l EDTA, pH 8 - 0, per litre H20). SOLUTIONS (1) Cell lysis buffer (5) Ficoll-Orange G 320 mmol/l sucrose. 0a1 % (w/v) Orange G in 20% (w/v) ficoll. A 10 mmol/l EDTA (pH 7 0). 0 I copyright. (6) Chloroform:octanol (24:1 v/v). (7) Restriction endonuclease buffer Prepare as a 10 x stock according to manufacturer's ring instructions. point 100n+160 mm lid (8) Denaturation solution http://jmg.bmj.com/ 0 5 mol/l NaOH. I1 5 mol/l NaCI. (9) Neutralisation buffer 0 5 mol/l Tris-HCI, pH 55. 3.0 mol/l NaCI. 0 3 mol/l sodium citrate. B on October 1, 2021 by guest. Protected (10) 20 x SSC 3 0 mol/l NaCl. 0]6 mm ]4mm 0 3 mol/l sodium citrate, pH 7 0. (11) 100 x Denhardt's 2% (w/v) bovine serum albumin fraction V. 2% (w/v) polyvinylpyrrolidone type 360. ring 2 % (w/v) Ficoll type 400. Heat to 400C while stirring, followed by gentle 24 mm stirring at 40C overnight. ]5mm (12) Hybridisation solution 40mm 3 x SSC. 100 mm 10 x Denhardt's. FIG 1 Diagram of the hybridisation chamber designed 0a 1% SDS (sodium dodecyl sulphate). by Alec Jeifreys showing the plane (A) and elevation (B). 10 ,ug/ml polyadenylic acid. J Med Genet: first published as 10.1136/jmg.21.3.164 on 1 June 1984. Downloaded from 166 Santie Vandenplas et al 50 ,ug/ml heat denatured sonicated herring sperm. of 500 V1 of a 5 mol/l sodium perchlorate solution DNA (see solution 14). was added. The digest was gently mixed with 8 ml of phenol :chloroform until homogeneous. The (13) Post-hybridisation wash solution were 3 x phases separated by centrifugation for 10 SSC. minutes at 12 000 at 100C. 10 x Denhardt's. g The upper, aqueous, phase was removed and further extracted with an 0-1 % sodium dodecyl sulphate. equal volume of chloroform:octanol (24:1). The (14) Heat denatured sonicated herring sperm DNA phases were again separated after gentle mixing. 2 mg/ml stock solution. DNA was precipitated from the aqueous phase by Dissolve and sonicate to an average length of adding 2 volumes of cold absolute ethanol. The approximately 600 base pairs. (Determine by agarose precipitate was lifted out with the sealed end of a gel electrophoresis. See Method section on agarose Pasteur pipette and shaken into 1 x TE. The DNA gel electrophoresis of DNA restriction fragments.) was allowed to dissolve overnight at 40C. A total of Denature in boiling waterbath for 10 minutes. Cool 0o 1 volumes 20 x SSC and 0 01 volumes 5 mg/ml rapidly on ice. ribonuclease were added and the mixture incubated for 1 hour at 370C. Then, 2 ml of sterile water was (15) Stringent wash solution added and the solution was extracted twice with 0-1 % (w/v) sodium dodecyl sulphate. chloroform:octanol (24:1). The DNA was pre- 0 1 x SSC (see discussion). cipitated by adding 2 volumes of absolute ethanol (16) 1 x TE and washed twice with 70% ethanol. The DNA 10 mm Tris-HCI, pH 7-5. pellet was dried under a vacuum for 15 minutes and 1 mmol/l EDTA. finally dissolved in 0 5 ml of sterile double distilled water. (17) 10 x T4 polymerase buffer The DNA concentration is estimated by the 330 mmol/l Tris-acetate, pH 7 9. determination of its absorbance at 260 nm assuming 660 mmol/l potassium acetate. that its Alm, 260 is 200 (that is, a 1 g/100 ml copyright. 100 mmol/l magnesium acetate. solution in a 1 cm lightpath has an absorbance at 1 mg/ml nuclease free bovine serum albumin. 260 nrm of 5 mmol/l dithiothreitol. 200).14 PREPARATION OF PROBE DNA The following stock solutions are also recommended: Details of the isolation of recombinant plasmids (a) 10% (w/v) sodium dodecyl sulphate. containing specific sequence probes are given by (b) 10 mg/ml proteinase K. Maniatis et al.12 Approval for these experiments by http://jmg.bmj.com/ (c) 10 mg/ml ribonuclease, heat treated at 800C for the Genetic Manipulation Advisory Group (GMAG) 10 minutes. is required. (d) 10 mg/ml ethidium bromide. (e) 0 1 mol/l EDTA, pH 7 0. Adjust pH in order to RESTRICTION ENDONUCLEASE DIGESTION dissolve EDTA. OF GENOMIC DNA (f) 5 mol/l sodium perchlorate. An incubation mixture with a final volume of 50 Vl1 was prepared containing 10 ,g DNA, 0 1 volume of Methods 10 x restriction endonuclease buffer, 30 units on October 1, 2021 by guest. Protected restriction enzymes (3 U/Vg DNA), and 100 ,ug/ml PREPARATION OF GENOMIC DNA nuclease-free bovine serum albumin.
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