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Denaturing high-performance liquid chromatography efficiently detects mutations of the acetolactate synthase gene

Published online by Cambridge University Press:  20 January 2017

Balazs Siminszky ,
Nicolas P. Coleman  and
Mariam Naveed
Nicolas P. Coleman
Affiliation:
Agronomy Department, University of Kentucky, Lexington, KY 40546-0312
Mariam Naveed
Affiliation:
Agronomy Department, University of Kentucky, Lexington, KY 40546-0312
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Abstract

Acetolactate synthase (ALS), a common enzyme in the biosynthesis of branched-chain amino acids, is a target for the sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinylthiobenzoate, and the sulfonylaminocarbonyltriazolinone classes of herbicides. Widespread resistance to the ALS-inhibiting herbicides has been attributed to single-base mutations in the ALS gene. The objective of this study was to investigate the feasibility of using denaturing high-performance liquid chromatography (DHPLC), a recently developed method of mutation analysis, for the detection of three ALS mutations, Ala122Thr, Leu574Trp, and Ser653Thr, which confer herbicide resistance. The mutated variants of the ALS gene were isolated from herbicide-resistant biotypes of smooth pigweed and Powell amaranth using polymerase chain reaction (PCR). The PCR products were hybridized with a wild-type sample and subjected to DHPLC analysis. All three mutations could be detected using a modified High performance liquid chromatography system; however, the sensitivity of the method was strongly dependent on the melting temperature profile of the analyzed DNA fragment. Once the primers and the DHPLC conditions are optimized, the procedure is economical, rapid, and requires little sample preparation. Because of these favorable features, DHPLC can be used as an alternative to other commonly used mutation detection methods.

Keywords

ImidazolinonepyrimidinylthiobenzoatesulfonylaminocarbonyltriazolinonesulfonylureatriazolopyrimidinePowell amaranth, Amaranthus powellii S. Wats. AMAPOsmooth pigweed, Amaranthus hybridus L. AMACHDenaturing high-performance liquid chromatographymutation detectionherbicide resistance
Type
Physiology, Chemistry, and Biochemistry
Information
Weed Science , Volume 53 , Issue 2 , April 2005 , pp. 146 - 152
Copyright
Copyright © Weed Science Society of America 

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References

Literature Cited

Devine, M. D. and Shukla, A. 2000. Altered target sites as a mechanism of herbicide resistance. Crop Prot 19:881–889.Google Scholar
Eng, C., Brody, L. C., and Wagner, T. M. U. et al. 2001. Interpreting epidemiological research: blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1. J. Med. Genet 38:824–833.CrossRefGoogle ScholarPubMed
Gerwick, B. C., Mireles, L. C., and Eilers, R. J. 1993. Rapid diagnosis of ALS/AHAS-resistant weeds. Weed Technol 7:519–524.Google Scholar
Haymes, K. M. 1996. Mini-prep method suitable for a plant breeding program. Plant Mol. Biol. Rep 14:280–284.CrossRefGoogle Scholar
Heap, I. M. 2004. The International Survey of Herbicide Resistant Weeds. www.weedscience.org.Google Scholar
Hecker, K. H., Asea, A., Kobayashi, K., Green, S., Tang, D., and Calderwood, S. K. 2000. Mutation detection in the human HSP70B gene by denaturing high-performance liquid chromatography. Cell Stress Chaperones 5:415–424.Google Scholar
Jones, A. C., Austin, J., Hansen, N., Hoogendoorn, B., Oefner, P. J., Cheadle, J. P., and O'Donovan, M. C. 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.Google Scholar
Kuk, Y. I., Jung, H. I., Kwon, O. D., Lee, D. J., Burgos, N. R., and Guh, J. O. 2003. Rapid diagnosis of resistance to sulfonylurea herbicides in monochoria (Monochoria vaginalis). Weed Sci 51:305–311.Google Scholar
Narayanaswami, G. and Taylor, P. D. 2001. Improved efficiency of mutation detection by denaturing high-performance liquid chromatography using modified primers and hybridization procedure. Genet. Test 5:9–16.CrossRefGoogle ScholarPubMed
Premstaller, A. and Oefner, P. J. 2002. Denaturing HPLC of nucleic acids. Lc Gc Europe 15:410.Google Scholar
Rees, W. A., Yager, T. D., Korte, J., and Vonhippel, P. H. 1993. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry 32:137–144.Google Scholar
Rozen, S. and Skaletsky, H. J. 2000. Primer3 on the WWW for general users and for biologist programmers. Pages 365–386 in Krawetz, S. and Misener, S. eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ: Humana.Google Scholar
Sanger, F., Nicklen, S., and Coulson, A. R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A 74:5463–5467.Google Scholar
Schloss, J. V. 1990. Acetolactate synthase, mechanism of action and its herbicide binding-site. Pestic. Sci 29:283–292.Google Scholar
Tranel, P. J. and Wright, T. R. 2002. Resistance of weeds to ALS-inhibiting herbicides: what have we learned? Weed Sci 50:700–712.Google Scholar
Wagner, T., Stoppa-Lyonnet, D., and Fleischmann, E. et al. 1999. Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations. Genomics 62:369–376.Google Scholar
Xiao, W. Z. and Oefner, P. J. 2001. Denaturing high-performance liquid chromatography: a review. Hum. Mutat 17:439–474.Google Scholar