Najd-Hassan-Bonab Leila, Hedayati Mehdi, Shahzadeh Fazeli Seyed Abolhassan, Daneshpour Maryam S
Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Molecular and Cellular Biology, Faculty of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, ACECR, Tehran, Iran.
Heliyon. 2023 Oct 19;9(11):e21102. doi: 10.1016/j.heliyon.2023.e21102. eCollection 2023 Nov.
Apolipoprotein E () is one of the most polymorphic genes at two single nucleotides (rs429358 and rs7412). The various isoforms of have been associated with a variety of diseases, including neurodegenerative, type 2 diabetes, etc. Hence, predicting the genotyping is critical for disease risk evaluation. The purpose of this study was to optimize the tetra amplification refractory mutation system (Tetra-ARMS) PCR method for the detection of APOE mutations.
Here, in our optimized Tetra-ARMS PCR method, different factors like cycle conditions, using HiFidelity enzyme instead of Taq polymerase and setting its best concentration, and the lack of using dimethylsulfoxide (DMSO) for amplifying the GC-regions were set up for all primer pairs. The sensitivity and accuracy were tested. For validation of the assay, the results were compared with known genotypes for the gene that were previously obtained by two independent methods, RFLP and Chip-typing.
Successful Tetra-ARMS PCR and genotyping are influenced by multiple factors. Our developed method enabled us to amplify the DNA fragment by 25 cycles without adding any hazardous reagent, like DMSO. Our findings showed 100 % accuracy and sensitivity of the optimized Tetra-ARMS PCR while both criteria were 95 % for RFLP and 100 % for the chip-typing method. In addition, our results showed 91 % and 100 % consistency with RFLP and chip typing methods, respectively.
Our current method is a simple and accurate approach for detecting polymorphisms within a large sample size in a short time and can be performed even in low-tech laboratories.
载脂蛋白E（APOE）是单核苷酸多态性最高的基因之一（rs429358和rs7412）。APOE的各种异构体与多种疾病相关，包括神经退行性疾病、2型糖尿病等。因此，预测APOE基因分型对于疾病风险评估至关重要。本研究的目的是优化四重扩增阻滞突变系统（Tetra-ARMS）PCR方法以检测APOE突变。
在我们优化的Tetra-ARMS PCR方法中，针对所有引物对设置了不同因素，如循环条件、使用高保真酶而非Taq聚合酶并设定其最佳浓度，以及在扩增GC区域时不使用二甲基亚砜（DMSO）。对灵敏度和准确性进行了测试。为验证该检测方法，将结果与先前通过两种独立方法（限制性片段长度多态性（RFLP）和芯片分型）获得的已知APOE基因分型进行比较。
成功的Tetra-ARMS PCR和基因分型受多种因素影响。我们开发的方法使我们能够在不添加任何有害试剂（如DMSO）的情况下通过25个循环扩增DNA片段。我们的研究结果显示，优化后的Tetra-ARMS PCR的准确性和灵敏度均为100%，而RFLP的这两个标准均为95%，芯片分型方法为100%。此外，我们的结果分别显示与RFLP和芯片分型方法的一致性为91%和100%。
我们目前的方法是一种简单准确的方法，可在短时间内对大量样本检测APOE多态性，甚至在技术水平较低的实验室也可进行。
