Chemical Cleavage of Heteroduplex DNA to Identify Mutations

Richard G. H. Cotton1, Markus Grompe2

1 Royal Children's Hospital, Parkville, 2 Oregon Health Sciences University, Portland, Oregon
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.6
DOI:  10.1002/0471142905.hg0706s17
Online Posting Date:  May, 2001
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Mutation screening by the chemical‐cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick‐paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild‐type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Chemical Cleavage of Heteroduplex DNA
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Chemical Cleavage of Heteroduplex DNA

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix
  • recipe10× annealing buffer (see recipe)
  • 100 to 150 ng unlabeled target mutant or test DNA, amplified by plasmid preps (unit 5.3 & CPMB UNITS & ) or PCR (unit 7.1)
  • 10 ng (∼1 × 105 cpm) radiolabeled probe ( appendix 3E)
  • 100% and 70% ethanol, ice cold
  • recipeHydroxylamine solution (see recipe)
  • recipe2.5× osmium tetroxide buffer (see recipe)
  • recipe4% (w/v) osmium tetroxide (see recipe)
  • recipeHOT stop buffer (see recipe)
  • 1 M piperidine (Fluka; prepare fresh)
  • 3 M sodium acetate, pH 5.2, containing 1.6 mg/ml tRNA
  • 2× formamide loading buffer ( appendix 2A)
  • Molecular weight marker: e.g., ϕX‐174 plasmid digested with HaeIII (Pharmacia Biotech or GIBCO/BRL) labeled with 32P or35S
  • 37°C, 65°C, 85°C, and 90°C water baths or heating block
  • 0.5‐ml microcentrifuge tubes, silanized (CPMB APPENDIX )
Additional reagents and equipment for nucleic acid quantitation ( appendix 3D) and preparation, electrophoresis, and drying of denaturing polyacrylamide gels ( appendix 3F)CAUTION: Radiolabeled probe, osmium tetroxide, piperidine, formamide loading buffer, and radiolabeled molecular weight markers are hazardous; see appendix 2A for guidelines handling, storage, and disposal.
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Literature Cited

Literature Cited
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   Bateman, J.F., Hannagan, M., Chan, D., and Cole, W.G. 1991. Characterization of a type I collagen α2(I) glycine‐586 to valine substitution in osteogenesis imperfect type IV. Biochem. J. 276:765‐770.
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   Cotton, R.G. and Campbell, R.D. 1989. Chemical reactivity of matched cytsine and thymine bases near mismatched and unmatched bases in a heteroduplex between DNA strands with multiple differences. Nucl. Acids Res. 17:4223‐4233.
   Cotton, R.G.H., Rodrigues, N.R., and Campbell, R.D. 1988. Reactivity of cytosine and thymine in single‐base‐pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc. Natl. Acad. Sci. U.S.A. 85:4397‐4401.
   Dianzani, I., Forrest, S.M., Camaschella, C., and Gottardi, E. 1991. Heterozygotes and homozygotes—discrimination by chemical cleavage of mismatch. Am. J. Hum. Genet. 48:423‐424.
   Forrest, S.M., Dahl, H.H., Howells, D.W., Dianzani, I., and Cotton, R.G.H. 1991. Mutation detection in phenylketonuria by using chemical cleavage of mismatch: Importance of using probes from both normal and patient samples. Am. J. Hum. Genet. 49:175‐183.
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   Maxam, A.M. and Gilbert, W. 1980. Sequencing end‐labeled DNA with base‐specific chemical cleavages. Methods Enzymol. 65:499‐560.
   Myers, R.M., Larin, Z., and Maniatis, T. 1985. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science 230:1242‐1246.
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   Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T. 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single‐strand conformation polymorphisms. Proc. Natl. Acad. Sci. U.S.A. 86:2766‐2770.
   Pilz, R.B., Steglich, C., and Scheffler, I.E. 1990. Molecular and genetic characterization of an ornithine decarboxylase‐deficient Chinese hamster cell line. J. Biol. Chem. 265:8880‐8886.
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   Saleeba, J.A., Ramus, S.J., and Cotton, R.G.H. 1992. Complete mutation detection using unlabeled chemical cleavage. Hum. Mut. 1:63‐69.
   Shapiro, J.R., Stover, M.L., Burn, V.E., and McKinstry, M.B. 1992. An osteopenic nonfracture syndrome with features of mild osteogenesis imperfecta associated with the substitution of a cysteine for glycine at triple helix position 43 in the proα1(I) chain of type I collagen. J. Clin. Invest. 89:567‐573.
   Sheffield, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M. 1989. Attachment of a 40‐base‐pair G+C‐rich sequence (GC‐clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single‐base changes. Proc. Natl. Acad. Sci. U.S.A. 86:232‐236.
   Theophilus, B.D.M., Latham, T., Grabowski, G.A., and Smith, F.I. 1989. Comparison of RNase A, a chemical cleavage and GC‐clamped denaturing gradient gel electrophoresis for the detection of mutations in exon 9 of the human acid‐glucosidase gene. Nucl. Acids Res. 17:7707‐7723.
   Winter, E., Yamamoto, F., Almoguera, C., and Perucho, M. 1985. A method to detect and characterize point mutations in the transcribed genes: Amplification and overexpression of the mutant c‐Ki‐ ras allele in human tumor cells. Proc. Natl. Acad. Sci. U.S.A. 82:7575‐7579.
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Key References
   Cotton et al., 1988. See above.
  Original description of the method applied to model mutations.
   Forrest et al., 1991. See above.
  Describes the application of the method using uniformly labeled probe generated during PCR.
   Grompe, M., Muzny, D.M., and Caskey, C.T. 1989. Scanning detection of mutations in human ornithine transcarboxylase (OTC) by chemical mismatch cleavage. Proc. Natl. Acad. Sci. U.S.A. 86:5888‐5892.
  Describes the application of the method using 5′ end‐labeled probe.
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