DHPLC Technology

DHPLC relies on the use of a hydrophobic column based on reverse-phase liquid chromatography for the separation of heteroduplex and homoduplex DNA at specific optimized temperatures. In DHPLC analysis, PCR amplicons are denatured and reannealed resulting in the formation of heteroduplex and homoduplex DNA. Homoduplex and heteroduplex DNA exhibit different melting properties allowing their separation during partially denaturing conditions (1). Heteroduplex DNA elutes before homoduplex DNA, both appearing as two or more peaks in the DHPLC chromatogram.

DHPLC can be used to find novel mutations and screen for known mutations. It is also useful in the detection of low percentage mutations in heterogeneous samples. Since its introduction in 1995 (2), DHPLC has gained tremendous popularity in the field of DNA mutation detection, especially over the last 5 years, owing to its high degree of automation and high sensitivity. In the study of mitochondrial DNA mutations, DHPLC has been used to either screen the whole mitochondrial genome for novel mutations (3-7) or study specific regions of the genome for known mutations (4,8-11), followed by DNA sequencing to identify the mutation sites.

 

References:

  1. Xiao, W. and Oefner, P.J. (2001) Denaturing high-performance liquid chromatography: a review. Hum. Mutat., 17, 439-474.
  2. Oefner, P.J. and Underhill, P.A. (1995) Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC). Am. J. Hum. Genet., 57, A266.
  3. Bayat, A., Walter, J., Lamb, H., Marino, M., Ferguson, M.W. and Ollier, W.E. (2005) Mitochondrial mutation detection using enhanced multiplex denaturing high-performance liquid chromatography. Int J Immunogenet, 32, 199-205.
  4. van Den Bosch, B.J., de Coo, R.F., Scholte, H.R., Nijland, J.G., van Den Bogaard, R., de Visser, M., de Die-Smulders, C.E. and Smeets, H.J. (2000) Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography.
    Nucleic Acids Res, 28, E89.
  5. Meierhofer, D., Mayr, J.A., Ebner, S., Sperl, W. and Kofler, B. (2005) Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations. Mitochondrion, 5, 282-296.
  6. Biggin, A., Henke, R., Bennetts, B., Thorburn, D.R. and Christodoulou, J. (2005) Mutation screening of the mitochondrial genome using denaturing high-performance liquid chromatography. Mol. Genet. Metab., 84, 61-74.
  7. Wulfert, M., Tapprich, C. and Gattermann, N. (2006) Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC.
    J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 831, 236-247.
  8. Liu, M.R., Pan, K.F., Li, Z.F., Wang, Y., Deng, D.J., Zhang, L. and Lu, Y.Y. (2002) Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography. World J Gastroenterol, 8, 426-430.
  9. Conley, Y.P., Brockway, H., Beatty, M. and Kerr, M.E. (2003) Qualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography. Brain Res Protoc, 12, 99-103.
  10. Lim, K.S., Naviaux, R.K. and Haas, R.H. (2007) Quantitative mitochondrial DNA mutation analysis by denaturing HPLC. Clin. Chem., 53, 1046-1052.
  11. Lim, K.S., Naviaux, R.K., Wong, S. and Haas, R.H. (2008) Pitfalls in the denaturing high-performance liquid chromatography analysis of mitochondrial DNA mutation. J. Mol. Diagn.,10, 102-108.