SpringerProtocols: Abstract: Mutation Scanning for the Clinical Laboratory: DHPLC

Mutation Scanning for the Clinical Laboratory: DHPLC

By: John F. Harvey³, Julian R. Sampson⁴

Abstract

Full Text

Download PDF (254K)

This chapter describes the Transgenomic WAVE® DNA Fragment Analysis System with DNA Sep® Technology, as used in our molecular genetics diagnostic laboratories for the detection of unknown mutations. Four software packages are currently available: WAVEmaker 3.4, 4, 4.1, and most recently, Navigator. Because versions 4 and 4.1 are similar, version 4 is presented here. Denaturing high-performance liquid chromatography (DHPLC), also known as TMHA (temperature-modulated heteroduplex analysis) provides a rapid, automated scanning method for mutations, whether known or unknown. Mutations are visualized as a characteristic pattern of peaks corresponding to the mixture of heteroduplexes and homoduplexes formed when wild-type and mutant DNA fragments are hybridized (1; Fig. 1).

MediaObjects/978-1-59259-432-0_3_Fig1_HTML.jpg

Fig. 1. Formation of a 1∶1 mixture of heteroduplexes and homoduplexes through hybridization.

Affiliation(s): (3) Wessex Regional Genetics Laboratory, Salisbury Health Care NHS Trust, Salisbury District Hospital, Salisbury, Wiltshire, UK
(4) Institute of Medical Genetics, University Hospital of Wales, Cardiff, UK

Book Title: Molecular Diagnosis of Genetic Diseases

Series: Methods in Molecular Medicine | Volume: 92 | Pub. Date: Oct-20-2003 | Page Range: 45-66 | DOI: 10.1385/1-59259-432-8:45

Subject: Genetics/Genomics

References

Download References

1.	Oefner, P. J. and Underhill, P. A. (1998) DNA mutation detection using denaturing high-performance liquid chromotography (DHPLC), in Current Protocols in Human Genetics (Dracopoli, N. C, Haines, J. L., Korf, B. R., Moir, D. T., Morton, C. C, Seidman, D. E., et al., eds.), 19, Wiley, New York, pp. 7.10.1–7.10.12.

2.	Haynes, S., Eccles, D., and Harvey, J. (2000) DHPLC analysis of HNPCC: a rapid sensitive exon screen of hMLH1 and hMSH2. J. Med. Genet. 37 (Suppl. 1), S42.

3.	Wagner, T., Stoppa-Lyonnet, D., Fleischmann, E., Muhr, D. I., Pages, S., Sandberg, T., et al. (1999) Denaturing high-performance liquid chromotography detects reliably BRCA1 and BRCA2 mutations. Genomics 62, 369–376.

4.	Gross, E., Arnold, N., Goette, J., Schwartz-Boeger, U., and Kiechle, M. (1999) A comparison of BRCA1 mutation analysis by direct sequencing. Hum. Genet. 105, 72–78.

5.	Giordano, M., Oefner, P. J., Underhill, P. A., Cavilli-Sforza, L. L., Tosi, R., and Richiardi, P. M. (1999) Identification by denaturing high-performance liquid chromatography of numerous polymorphisms in a candidate region for multiple sclerosis susceptibility. Genomics 56, 247–253.

6.	Liu, W. L., Oefner, P. J., Qian, C, Odom, R. S., and Francke, U. (1998) Denaturing HPLC-identified novel FBN1 mutations, polymorphisms, and sequence variants in Marfan syndrome and related connective tissue disorders. Genet. Test. 1, 237–242.

7.	Jones, A. C, Sampson, J. R., Hoogendoorn, B., Cohen, D., and Cheadle, J. P. (2000) Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis. Hum. Genet. 106, 663–668.

8.	Klein, B., Weirich, G., and Brauch, H. (2001) DHPLC-based germline mutation screening in the analysis of the VHL tumor suppressor gene: usefulness and limitations. Hum. Genet. 108, 376–384.

9.	Holinksi-Feder, E., Mller-Koch, Y., Friedl, W., Moeslain, G, Keller, G, Plaschke, J., et al. (2001) DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2. J. Biochem. Biophys. Methods 47, 21–32.

10.	Jones, A. C, Sampson, J. R., and Cheadle, J. P. (2001) Low level mosaicism detectable by DHPLC but not by direct sequencing. Hum. Mutat. 17, 233–234.

11.	Donohue, E. and Baty, D. U. (2000) The use of DHPLC technology to detect PLP gene mutations in Pelizaeus-Merzbacher patients. J. Med. Genet. 37 (Suppl. 1), S77.

12.	Hoogendoorn, B., Norton, N., Kirov, G., Williams, N., Hamshere, M. L., Spurlock, G., et al. (2000) Cheap, accurate and rapid allele frequency estimation of single nucleotide polymorphisms by primer extension and DHPLC in DNA pools. Hum. Genet. 107, 488–493.

13.	CMGS Best Practice Guidelines for Use of the WAVE System in Diagnostic Service: http://www.CMGS.org/BPG/Guidelines/2002/dhplc.htm.

Comments (Loading...)

Post comment/View all comments