Abstract
Since its introduction more than a decade ago, denaturing HPLC has been widely used to screen nuclear and mitochondrial DNA for mutations. We recently developed a quantitative method based on denaturing HPLC to measure DNA mutation load, using tRNA Leu(UUR) region of the mitochondrial DNA as an example. The mutation load is determined based on the quadratic function that governs DNA reannealing and the formation of heteroduplex and homoduplex DNAs. We have used this assay to measure heteroplasmy level for several mitochondrial mutations in DNAs obtained from human tissue samples. This quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Underhill, P. A., Jin, L., Lin, A. A., et al. (1997) Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography. Genome Res. 7, 996–1005.
O'Donovan, M. C., Oefner, P. J., Roberts, S. C., et al. (1998) Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics 52, 44–49.
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. Nucleic Acids Res. 26, 1396–1400.
Jones, A. C., Austin, J., Hansen, N., et al. (1999) Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clin. Chem. 45, 1133–1140.
Wagner, T., Stoppa-Lyonnet, D., Fleischmann, E., et al. (1999) Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations. Genomics 62, 369–376.
Gross, E., Arnold, N., Pfeifer, K., Bandick, K., and Kiechle, M. (2000) Identification of specific BRCA1 and BRCA2 variants by DHPLC. Hum. Mutat. 16, 345–353.
van Den Bosch, B. J., de Coo, R. F., Scholte, H. R., et al. (2000) Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography. Nucleic Acids Res. 28, E89.
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.
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.
Xiao, W., and Oefner, P. J. (2001) Denaturing high-performance liquid chromatography: a review. Hum. Mutat. 17, 439–474.
Oefner, P. J., and Huber, C. G. (2002) A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782, 27–55.
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.
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.
Liu, M. R., Pan, K. F., Li, Z. F., et al. (2002) Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography. World J. Gastroenterol. 8, 426–430.
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.
Lim, K. S., Naviaux, R. K., and Haas, R. H. (2007) Quantitative mitochondrial DNA mutation analysis by denaturing HPLC. Clin. Chem. 53, 1046–1052.
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.
Su, Y. N., Lee, C. N., Hung, C. C., et al. (2003) Rapid detection of beta-globin gene (HBB) mutations coupling heteroduplex and primer-extension analysis by DHPLC. Hum. Mutat. 22, 326–336.
Nickerson, M. L., Warren, M. B., Zbar, B., and Schmidt, L. S. (2001) Random mutagenesis-PCR to introduce alterations into defined DNA sequences for validation of SNP and mutation detection methods. Hum. Mutat. 17, 210–219.
Nickerson, M. L., Weirich, G., Zbar, B., and Schmidt, L. S. (2000) Signature-based analysis of MET proto-oncogene mutations using DHPLC. Hum. Mutat. 16, 68–76.
Ezzeldin, H., Okamoto, Y., Johnson, M. R., and Diasio, R. B. (2002) A high-throughput denaturing high-performance liquid chromatography method for the identification of variant alleles associated with dihydropyrimidine dehydrogenase deficiency. Anal. Biochem. 306, 63–73.
Bayat, A., Walter, J. M., Lamb, H., Ferguson, M. W., and Ollier, W. E. (2003) Rapid denaturing high-performance liquid chromatography (DHPLC) for mutation scanning of the transforming growth factor beta3 gene using a novel proof-reading polymerase. Eur. J. Immunogenet. 30, 335–340.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lim, K.S., Naviaux, R.K., Haas, R.H. (2009). Determination of DNA Mutation Load in Human Tissues Using Denaturing HPLC-Based Heteroduplex Analysis. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_18
Download citation
DOI: https://doi.org/10.1007/978-1-59745-521-3_18
Publisher Name: Humana Press
Print ISBN: 978-1-934115-60-2
Online ISBN: 978-1-59745-521-3
eBook Packages: Springer Protocols