Program in Molecular Medicine
Alanine; Amino Acid Substitution; Arabidopsis; Arabidopsis Proteins; Base Sequence; DNA Mutational Analysis; Deoxyribonucleases; *Disease; Factor V; Gene Expression Regulation, Enzymologic; Genome; Glycine; HIV-1; Humans; Molecular Sequence Data; Mutagenesis; Mutation; Peas; Venous Thrombosis
Biochemistry, Biophysics, and Structural Biology | Life Sciences | Medicine and Health Sciences
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.
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© 2008 Triques et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
DOI of Published Version
BMC Mol Biol. 2008 Apr 23;9:42. Link to article on publisher's site
BMC molecular biology
Triques, Karine; Piednoir, Elodie; Dalmais, Marion; Schmidt, Julien; Le Signor, Christine; Sharkey, Mark E.; Caboche, Michel; Sturbois, Benedicte; and Bendahmane, Abdelhafid, "Mutation Detection Using ENDO1: Application to Disease Diagnostics in Humans and TILLING and Eco-TILLING in Plants" (2008). Open Access Articles. 2293.