User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

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

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

¹Department of Molecular and Clinical Genetics, Central Clinical School and SUPAMAC The University of Sydney, New South Wales, Australia
²SUPAMAC, The University of Sydney, New South Wales, Australia
³Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, New South Wales, Australia
⁴Stanford 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

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

Learn more about the author(s):

Bing Yu

Abstract

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Basic Protocol
Reagents and Solutions
Commentary
Literature Cited
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol

Materials

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 MgCl₂
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., 1992)
PCR-quality deionized water (e.g., 18 M 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 15.1), agarose gel electrophoresis and ethidium bromide staining (unit 2.7), and mutation detection by DNA sequencing (unit 7.7 and cpmb Chapter 7)

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 7.10.1

Elevated column temperature causes partial denaturation and consequent separation of the hetero- and homoduplex species that constitute the sample. Heteroduplex species are generally retained less, due to their decreased interaction with the ion pairing reagent (triethylammonium ion). Further resolution of the hetero- and homoduplex species is not always observed. In the present case, separation has been attained, most likely due to differences in neighboring stacking interactions (Xiao and Oefner, 2001), yielding the following order of stabilities: AC < GT < AT < GC. Data were obtained using the following equipment and conditions. Equipment: Varian chromatograph consisting of two SD-200 pumps; model AI-1A automatic sample injector; CH-1 column oven; UV-C absorbance detector; Macintosh-based method management and data acquisition; and 50 mm × 4.6-mm-i.d. DNASep column (Transgenomic). Conditions: buffer A composition, 0.1 M TEAA, pH 7/0.1 mM tetrasodium EDTA; and buffer B composition 0.1 M TEAA, pH 7/25% acetonitrile; gradient of 50% to 54.5% buffer B in 0.5 min, followed by 54.5% to 60% B in 3.0 min, 95% B for 0.7 min, and 50% B for 1.0 min; flow rate 0.9 ml/min; and temperature 56°C.

View Image
Figure 7.10.2

Impact of column temperature on the resolution of homo- and heteroduplex molecules of a 209-bp amplicon containing a single AG transition. Data obtained using the following equipment and conditions. Equipment: Wave Nucleic Acid Fragment Analysis System Model 3500HT; DNASep HT Cartridge (37 mm × 6.5 mm-i.d., Transgenomic); “Rapid DNA Analysis” procotol; and injection volume of 5 µl. Similar chromatographic profiles over 6 to 7 min, showing an identical temperature range over which the mismatch can be detected, have been obtained with Varian Helix or HP 1100 Series system (Xiao and Oefner, 2001). The best temperature in this example is 56°C. The ability of DHPLC to detect mutations over an extended temperature range explains its high sensitivity.

View Image
Figure 7.10.3

Multiplex analysis of three different amplicons in a single DHPLC run. Samples were amplified, denatured, and reannealed separately before being combined and analyzed. Data obtained using the chromatograph and column as described in Figure 7.10.1, and the following conditions: buffer A composition 0.1 M TEAA, pH 7/0.1 mM tetrasodium EDTA; and buffer B composition 0.1 M TEAA, pH 7/25% acetonitrile; gradient of 50% to 56% buffer B in 0.5 min, followed by 56% to 68.6% B in 7.0 min, 95% B for 0.7 min, and 50% B for 1.0 min; flow rate 0.9 ml/min; temperature 55°C. Sample identification: (A) three haploid Y chromosome amplicons of an African reference male of 422, 391, and 570 bp in size, (B) reference amplicons hybridized in order of elution to an Australian aboriginal, Chinese, and New Guinean Y chromosome that differed from the reference in a CT transition, a 2-bp deletion, and an AG transition, respectively. Note that the 422-bp amplicon eluted earlier than the 391-bp amplicon because of greater AT content and resulting more extensive partial denaturation.

View Image

Literature Cited

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
	http://www.ncbi.nlm.nih.gov/entrez/query.fcgi db=OMIM
Online Mendelian Inheritance in Man.
	http://insertion.stanford.edu/melt.html
DHPLC Analysis Temperature Prediction.
	http://www.transgenomic.com/
	http://www.varianinc.com/
	http://www.chem.agilent.com/cag/peak/peak3-95/introducing.html
DHPLC Instruments.
	http://hplc.chem.shu.edu/BOOK/xfram.html
	http://www.transgenomic.com/lib/JournalArticles.asp
	http://insertion.stanford.edu/pub.html
DHPLC references.
	http://www.cmgs.org/BPG/Guidelines/2002/dhplc.htm
	http://www.mutationdiscovery.com/
DHPLC Analysis Guidelines.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Author Notes

Submitted by: Bing Yu

On: June 15, 2009

DHPLC is also a useful tool for the detection and characterisation of somatic changes, heteroplasmic mutations or mosaicism. The heterduplex peak(s) can be defined and recovered through the DHPLC fraction collector. Consequently the variant component will be enriched in term of its proportion to the wild-type allele. The recovered solution can be re-amplified for further sequencing characterisation. Although the high resolution melting method can also detect the above variants in some cases, it cannot further characterise the identified variants. We demonstrated this potential of DHPLC in a recent publication (Luquin N, et al. Amyotroph Lateral Scler. 2008 Nov 26:1-7. [Epub ahead of print, PMID: 19034747]).

Submitted by: Bing Yu

On: June 15, 2009