College of Health Sciences, Korea University, Seoul.
J Toxicol Environ Health A. 2010;73(21-22):1502-10. doi: 10.1080/15287394.2010.511573.
Polymerase chain reaction (PCR) is a powerful molecular biological tool in the field of toxicity testing and diagnostics. The use of PCR for large-scale genetic testing requires an effective method of sample processing. Unfortunately, isolation of PCR-quality DNA is time-consuming. PCR performed directly on whole blood is preferred because of time efficiency, cost of the procedure, and possible automation for large-scale toxicity evaluation and diagnosis. The apolipoprotein E (APOE) gene contains two single-nucleotide polymorphisms (SNP) located at codons 112 and 158, producing three APOE protein isoforms known to be associated with the risks of developing cardiovascular disease and susceptibility to Alzheimer's disease. In the present study, an attempt was made to use the AnyDirect solution for APOE genotyping by PCR using whole blood directly without DNA purification. Results for two PCR methods, (1) conventional PCR using purified DNA and conventional buffer and (2) direct PCR using whole blood and AnyDirect solution, were compared in four different PCR-based APOE genotyping methods including PCR restriction-fragment-length polymorphism (PCR-RFLP), allele-specific PCR, SNaPshot mini-sequencing, and multiplex tetra-primer amplification refractory mutation system (T-ARMS) PCR. There was complete concordance in the APOE genotypes between conventional PCR and direct PCR, in all four different PCR-based APOE genotyping methods. Data demonstrated that the four different PCR-based APOE genotyping methods are able to determine the APOE genotypes successfully using whole blood directly with the use of AnyDirect solution. The direct multiplex T-ARMS PCR using whole blood may be the most rapid, simple, and inexpensive method for detecting APOE genotypes among four different APOE genotyping methods.
聚合酶链式反应（PCR）是毒性测试和诊断领域的一种强大的分子生物学工具。PCR 用于大规模基因测试需要有效的样品处理方法。不幸的是，PCR 质量的 DNA 分离既耗时又费力。直接从全血中进行 PCR 因其时间效率、程序成本以及可能实现大规模毒性评估和诊断的自动化而受到青睐。载脂蛋白 E（APOE）基因包含两个位于密码子 112 和 158 的单核苷酸多态性（SNP），产生三种 APOE 蛋白同工型，已知与心血管疾病风险和阿尔茨海默病易感性相关。在本研究中，尝试使用 AnyDirect 溶液直接从全血中进行 PCR 而无需 DNA 纯化，从而对 APOE 进行基因分型。比较了两种 PCR 方法的结果，（1）使用纯化 DNA 和常规缓冲液的常规 PCR，（2）使用全血和 AnyDirect 溶液的直接 PCR，在包括 PCR 限制性片段长度多态性（PCR-RFLP）、等位基因特异性 PCR、SNaPshot 迷你测序和多重四引物扩增不可抑制突变系统（T-ARMS）PCR 在内的四种不同的基于 PCR 的 APOE 基因分型方法中。在所有四种不同的基于 PCR 的 APOE 基因分型方法中，常规 PCR 和直接 PCR 的 APOE 基因型完全一致。数据表明，四种不同的基于 PCR 的 APOE 基因分型方法能够成功地直接使用全血和 AnyDirect 溶液确定 APOE 基因型。直接的多重 T-ARMS PCR 使用全血可能是四种不同的 APOE 基因分型方法中最快速、简单和经济的检测 APOE 基因型的方法。
