DNA Mutation Detection Using Denaturing High‐Performance Liquid Chromatography (DHPLC)

Bing Yu1, Nicole A. Sawyer2, Christine Chiu3, Peter J. Oefner4, Peter A. Underhill4

1 Department of Molecular and Clinical Genetics, Central Clinical School and SUPAMAC The University of Sydney, New South Wales, 2 SUPAMAC, The University of Sydney, New South Wales, 3 Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, New South Wales, 4 Stanford University, Pasadena, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.10
DOI:  10.1002/0471142905.hg0710s48
Online Posting Date:  February, 2006
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DHPLC is an efficient method for candidate gene scanning with a high level of automation. Single‐base substitutions and insertions or deletions of up to 1.5 kb in PCR amplified DNA fragments can be detected. The method exploits the differential retention of homoduplex and heteroduplex DNA species under conditions of partial thermal denaturation. DHPLC provides a useful platform for high‐throughput mutation detection and SNP discovery.

Keywords: denaturing high performance liquid chromatography; mutation detection; DNA heteroduplex; temperature‐modulated fractionation; high‐throughput

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Genomic DNA (of concentration such that the PCR reaction will contain 0.5 to 1 ng/µl template DNA), preferably isolated by high‐salt precipitation (see unit 7.1), including samples to be tested for a polymorphism or mutation and positive controls, if available, known to be heterozygous and homozygous for that genetic variant
  • 10× GeneAmp PCR buffer II (Applied Biosystems) or equivalent (500 mM KCl/100 mM Tris⋅Cl, pH 8.3)
  • 25 mM MgCl 2
  • 2.5 to 5 mM solutions of each dNTP (dCTP, dATP, dTTP, dGTP; appendix 2D)
  • 20 pmol/µl each of forward and reverse amplification primers (see recipe for PCR primers)
  • 5 U/µl AmpliTaq Gold DNA polymerase (Applied Biosystems) or standard (preferably high‐fidelity) Taq DNA polymerase in conjunction with alternative “hot‐start” scheme (Chou et al., )
  • PCR‐quality deionized water (e.g., 18 MΩ Milli‐Q water; do not autoclave)
  • DHPLC buffers A and B (see recipe); alternatively, purchase from commercial suppliers, e.g., Wave Optimized Buffers (Transgenomic) or Helix BufferPaks (Varian)
  • HaeIII restriction digest of pUC18 DNA (Transgenomic, Varian, or Sigma) or other commercially available plasmid digest for quality‐control purposes
  • PCR tubes
  • Thermal cycler (e.g., Applied Biosystems GenAmp PCR System 9700 or equivalent)
  • HPLC instrumentation including precolumn filter, vacuum degasser, oven, and UV or fluorescent detector: e.g., Wave Nucleic Acid Fragment Analysis System (Transgenomic), Helix System (Varian), or HP 1100 Series HPLC System (Agilent)
  • DHPLC analysis columns:
    • For Transgenomic system: DNASep Cartridge, DNASep HT Cartridge, DNASep Prep Cartridge, OligoSep Cartridge, OligoSep Prep HC Cartridge, or and RNASep Prep Cartridge
    • For Varian system: Helix DNA Column
    • For Agilent system: Eclipse dsDNA Analysis Column
  • Additional reagents and equipment for preparation of genomic DNA (unit 7.1), PCR (CPMB UNIT ), agarose gel electrophoresis and ethidium bromide staining (unit 2.7), and mutation detection by DNA sequencing (unit 7.7 and CPMB Chapter 7)
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Literature Cited

   Chou, Q., Russell, M., Birch, D.E., Raymond, J., and Bloch, W. 1992. Prevention of pre‐PCR mis‐priming and primer dimerization improves low‐copy‐number amplifications. Nucl. Acids Res. 20:1717‐1723.
   Dierick, H., Stul, M., DeKilver, W., Marynen, P., and Cassiman, J.J. 1993. Incorporation of dITP or 7‐deaza‐dGTP during PCR improves sequencing of the product. Nucl. Acids Res. 21:4427‐4428.
   Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K., and Mattick, J.S. 1991. “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucl. Acids Res. 19:4008.
   Eng, C., Brody, L.C., Wagner, T.M., Devilee, P., Vijg, J., Szabo, C., Tavtigian, S.V., Nathanson, K.L., Ostrander, E., and Frank, T.S., Steering Committee of the Breast Cancer Information Core (BIC) Consortium. 2001. Interpreting epidemiological research: Blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1. J. Med. Genet. 38:824‐833.
   Hayward‐Lester, A., Chilton, B.S., Underhill, P.A., Oefner, P.J., and Doris, P.A. 1997. Quantification of specific nucleic acids, regulated RNA processing and genomic polymorphisms using reversed‐phase HPLC. In Gene Quantification (F. Ferre, ed.) pp. 44‐77. Birkhauser Verlag, Basel, Switzerland.
   Huber, C.G., Oefner, P.J., and Bonn, G.K. 1993. High‐resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene‐divinylbenzene copolymers. Anal. Biochem. 212:351‐358.
   Huber, C.G., Oefner, P.J., and Bonn, G.K. 1995. Rapid and accurate sizing of DNA fragments by ion‐pair chromatography on alkylated nonporous poly(styrene‐divinylbenzene) particles. Anal. Chem. 67:578‐585.
   Innis, M.A. and Gelfand, D.H. 1990. Optimization of PCRs. In PCR Protocols: A Guide to Methods and Applications (M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White, eds.) pp. 3‐12. Academic Press, San Diego.
   Levinson, G. and Gutman, G.A. 1987. Slipped‐strand mispairing: A major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4:203‐221.
   Liu, W., Smith, D.I., Rechtzigel, K.J., Thibodeau, S.N., and James, C.D. 1998. Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucl. Acids Res. 26:1396‐1400.
   McDowell, D.G., Burns, N.A., and Parkes, H.C. 1998. Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR. Nucl. Acids Res. 26:3340‐3347.
   O'Donovan, M.C., Oefner, P.J., Austin, J., Hoogendoorn, B., Guy, C., Speight, G., Upadhyaya, M., Sommer, S.S., and McGuffin, P. 1998. Blind analysis of denaturing high performance liquid chromatography as a tool for mutation detection. Genomics 52:44‐49.
   Oefner, P.J., Huber, C.G., Umlauft, F., Berti, G.N., Stimpfl, E., and Bonn, G.K. 1994. High‐resolution liquid chromatography of fluorescent dye‐labeled nucleic acids. Anal. Biochem. 223:39‐46.
   Ophoff, R.A., Terwindt, G.M., Vergouwe, M.N., van Eijk, R., Oefner, P.J., Hoffman, S.M.G., Lamerdin, J.E., Mohrenweiser, H.W., Bulman, D.E., Ferrari, M., Haan, J., Lindhout, D., van Ommen, G.J., Hofker, M.H., Ferrari, M.D., and Frants, R.R. 1996. Familial hemiplegic migraine and episodic ataxia type‐2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 87:543‐552.
   Petrij‐Bosch, A., Peelen, T., van Vliet, M., van Eijk, R., Olmer, R., Drusedau, M., Hogervorst, F.B.L., Hageman, S., Arts, P.J.W., Ligtenberg, M.J.L., Meijers‐Heijboer, H., Klijn, J.G.M., Vasen, H.F.A., Cornelisse, C.J., van't Veer, L.J., Bakker, E., van Ommen, G‐J.B., and Devilee, P. 1997. BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients. Nat. Genet. 17:341‐345.
   Prince, A.M. and Andrus, L. 1992. PCR: How to kill unwanted DNA. BioTechniques 12:358‐360.
   Ravnik‐Glavac, M., Atkinson, A., Glavac, D., and Dean, M. 2002. DHPLC screening of cystic fibrosis gene mutations. Hum. Mutat. 19:374‐383.
   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.
   Spiegelman, J.I., Mindrinos, M.N., and Oefner, P.J. 2000. High‐accuracy DNA sequence variation screening by DHPLC. BioTechniques 29:1084‐1090.
   Turner, S.L. and Jenkins, F.J. 1995. Use of deoxyinosine in PCR to improve amplification of GC‐rich DNA. BioTechniques 19:48‐52.
   Underhill, P.A., Jin, L., Lin, A.L., Mehdi, S.Q., Jenkins, T., Vollrath, D., Davis, R., Cavalli‐Sforza, L.L., and Oefner, P.J. 1997. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high performance liquid chromatography. Genome Res. 7:996‐1005.
   Wong, K.K. and McClelland, M. 1991. PCR with 5‐methyl‐dCTP replacing dCTP. Nucl. Acids Res. 19:1081‐1085.
   Wong, K.K., Markillie, L.M., and Saffer, J.D. 1997. A novel method for producing partial restriction digestion of DNA fragments by PCR with 5‐methyl‐CTP. Nucl. Acids Res. 25:4169‐4171.
   Xiao, W. and Oefner, P.J. 2001. Denaturing high‐performance liquid chromatography: A review. Hum. Mutat. 17:439‐474.
   Xiao, W., Stern, D., Jain, M., Huber, C.G., and Oefner, P.J. 2001. Multiplex capillary denaturing high‐performance liquid chromatography with laser‐induced fluorescence detection. BioTechniques 30:1332‐1338.
   Yu, B., Sawyer, N.A., Caramins, M., Yuan, Z.G., Saunderson, R.B., Pamphlett, R., Richmond, D.R., Jeremy, R.W., and Trent, R.J. 2005. Denaturing high performance liquid chromatography: High throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease. J. Clin. Pathol. 58:479‐485.
Key References
   Liu et al., 1998. See above.
  Examples of DHPLC mutation detection in both somatic and tumor tissue samples.
   Xiao and Oefner, 2001. See above.
  Overview of DHPLC analysis mechanism, development of instruments, and various applications.
Internet Resources
  Online Mendelian Inheritance in Man.
  DHPLC Analysis Temperature Prediction.
  DHPLC Instruments.
  DHPLC references.
  DHPLC Analysis Guidelines.
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