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


CONCLUSIONS
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.
Triques
Karine
K
Unité Mixte de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, CP5708, 91 057 Evry Cedex, France. karine.triques@interieur.gouv.fr
Piednoir
Elodie
E
Dalmais
Marion
M
Schmidt
Julien
J
Le Signor
Christine
C
Sharkey
Mark
M
Caboche
Michel
M
Sturbois
Bénédicte
B
Bendahmane
Abdelhafid
A
D000409:Alanine; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004194:Disease; D005165:Factor V / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D016678:Genome; D005998:Glycine / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D018532:Pisum sativum / Q000235:genetics*; D020246:Venous Thrombosis / Q000175:diagnosis / Q000235:genetics
D029681:Arabidopsis Proteins; C513622:BFN1 protein, Arabidopsis; D005165:Factor V; D003851:Deoxyribonucleases; D000409:Alanine; D005998:Glycine