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

Karine Triques,Julien Schmidt,Elodie Piednoir,Bénédicte Sturbois,Michel Caboche,Mark Sharkey,Abdelhafid Bendahmane,Marion Dalmais,Christine Le Signor

doi:10.1186/1471-2199-9-42

Karine Triques, Julien Schmidt + Show 7 more

Open Access

https://doi.org/10.1186/1471-2199-9-42

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Abstract

BackgroundMost 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.ResultsThe 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.ConclusionWe 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.

Highlights

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
The construct was transformed into Agrobacterium and co-agroinfiltrated into N. benthamiana leaves in the presence of the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of post-transcriptional gene silencing (PTGS) in infiltrated tissues and allows a high level of transient expression (Figure 1a) [32]
Protein extract from leaves co-agroinfiltrated with TBSV p19 and green fluorescent protein (GFP), instead of ENDO1 construct, was used as a negative control

Summary

Introduction

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. BMC Molecular Biology 2008, 9:42 http://www.biomedcentral.com/1471-2199/9/42 eral mutation detection techniques based on the physical properties of single stranded DNA or heteroduplex DNA [1,2,3,4,5] have been described Among such methods are conformation sensitive gel electrophoresis (CSGE) [3], denaturing gradient gel electrophoresis (DGGE) [6], constant denaturing capillary electrophoresis, (CDCE) [7], Temperature Gradient Capillary Electrophoresis (TGCE) [8], single strand conformation polymorphism (SSCP) [2] and denaturing high-performance liquid chromatography (DHPLC) [1]. Many mismatch specific cleavage enzymes were reported to cleave preferentially certain types of mismatches, display low sensitivity or lead to a high background [10,11,20,28,29]

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