Triques Karine, Piednoir Elodie, Dalmais Marion, Schmidt Julien, Le Signor Christine, Sharkey Mark, Caboche Michel, Sturbois Bénédicte, Bendahmane Abdelhafid
Unité Mixte de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, CP5708, 91 057 Evry Cedex, France.
BMC Mol Biol. 2008 Apr 23;9:42. doi: 10.1186/1471-2199-9-42.
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.
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.
大多数酶促突变检测方法基于错配特异性核酸内切酶在错配位点切割异源双链DNA，并在DNA测序仪上分析消化产物。这些方法的重要局限性包括错配特异性核酸内切酶的可用性、它们在检测DNA池中一个等位基因时的灵敏度、分析成本以及该技术在标准分子生物学实验室中实施的难易程度。
将ENDO1和p19构建体共农杆菌渗入本氏烟草叶片，可实现来自拟南芥的错配特异性且灵敏的核酸内切酶ENDO1的高水平瞬时表达。我们展示了所产生的酶在突变检测中的广泛用途。在人类中，我们报告了与静脉血栓形成相关的莱顿因子V基因中G1691A突变的诊断以及接受抗逆转录病毒治疗患者中HIV-1准种的指纹图谱分析。在植物中，我们报告了使用ENDO1系统通过豌豆中的定向诱导基因组局部突变（TILLING）检测视网膜母细胞瘤相关基因突变等位基因，并通过拟南芥中的生态TILLING发现自然序列变异。
我们基于错配特异性且灵敏的核酸内切酶ENDO1的简化纯化方案引入了一种经济高效的工具。特别是，我们报告了ENDO1在人类突变诊断、复杂病毒群体指纹图谱分析以及植物TILLING和生态TILLING中的成功应用。
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