Mutation detection using ENDO1: Application to disease diagnostics in humans and TILLING and Eco-TILLING in plants

Karine Triques¹ , Elodie Piednoir¹ , Marion Dalmais¹ , Julien Schmidt¹ , Christine Le Signor³ , Mark Sharkey² , Michel Caboche¹ , Bénédicte Sturbois¹ and Abdelhafid Bendahmane¹

¹Unité Mixte de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, CP5708, 91 057 Evry Cedex, France

²University of Massachusetts Medical School, Worcester, Massachusetts, USA

³Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses, Domaine d'Epoisses, 21110 Bretenières, France

author email corresponding author email

BMC Molecular Biology 2008, 9:42doi:10.1186/1471-2199-9-42


Published:	23 April 2008

Abstract

Background

Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory.

Results

The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana.

Conclusion

We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.