von Ahsen N, Oellerich M, Armstrong V W, Schütz E
Department of Clinical Chemistry, Georg-August-University, Robert-Koch-Strasse 40, 37075 Goettingen, Germany.
Clin Chem. 1999 Dec;45(12):2094-101.
PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations.
We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented.
All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; S(y|x) = 0.96; n = 24).
This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.
基于聚合酶链反应（PCR）的突变检测容易出现方法学错误，例如，在限制性片段长度多态性（RFLP）和等位基因特异性扩增（ASA）中，由于技术故障或非典型新突变可能会出现假PCR结果。
我们研究了基于标记寡核苷酸杂交的基因分型检测方法检测和区分凝血因子V和载脂蛋白B - 100基因中已知和未知突变的能力。使用核酸双链稳定性的最近邻模型计算预期熔解温度，并与LightCycler熔解曲线得出的实验结果进行比较。提出了一种对载脂蛋白B - 100基因G10699A和C10698T突变进行基因分型的方法。
使用单个探针可以检测到所探测序列中测试的所有错配。测量的熔解温度与使用最近邻模型预测的值高度一致（r = 0.96；y = 0.98x + 1.18；S(y|x) = 0.96；n = 24）。
该方法不仅能够识别感兴趣的突变，还能将其与前者附近的其他潜在突变区分开来。最近邻模型对LightCycler上的杂交探针检测有效，在建立此类检测中应具有普遍价值。我们通过两个临床相关的基因分型实例表明，与传统的基于RFLP或ASA PCR的分子诊断方法相比，LightCycler突变检测系统具有卓越的区分性能。使用这种方法，在每个杂交探针检测中，在设计合理的探针下所有突变都应可检测到，但在所有情况下不一定能将它们彼此区分开来。
