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

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.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.Scanning DNA sequences for mutations and polymorphisms is an analytic tool in a broad range of disciplines. However, identification of such mutations and polymorphisms in long stretches of DNA and in large numbers of samples by direct sequencing is not a trivial exercise. Several mutation detection techniques based on the physical properties of single stranded DNA or heteroduplex DNA [1-5] 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]. Other methods exploit chemicals like groove binders or chemicals that cleave single strand DNA at the mismatch site in heteroduplex DNA [9].Single strand specific nucleases