一种用于同时检测马来西亚马来人 MTHFR 677 C > T、eNOS +894 G > T 和 -eNOS -786 T > C 变异的新型多重 PCR-RFLP 方法。
A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays.
机构信息
Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
出版信息
BMC Med Genet. 2012 May 17;13:34. doi: 10.1186/1471-2350-13-34.
BACKGROUND
Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS -786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C were calculated using the Hardy Weinberg equation.
METHODS
The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.
RESULTS
The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS -786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).
CONCLUSIONS
Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants.
背景
亚甲基四氢叶酸还原酶（MTHFR）677 C>T 变体导致的高同型半胱氨酸血症与心血管疾病和中风有关。另一个可能导致这些疾病的因素是一氧化氮水平降低，这可能是由于内皮型一氧化氮合酶（eNOS）+894 G>T 和 eNOS-786 T>C 变体的存在，使个体更容易发生内皮功能障碍。已经开发了许多基因分型方法来研究这些变体。然而，使用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）分析的同时检测方法仍然缺乏。在这项研究中，开发了一种新的用于同时检测 MTHFR 677 C>T 和 eNOS +894 G>T 和 eNOS -786 T>C 变体的多重 PCR-RFLP 方法。共招募了 114 名健康的马来人作为研究对象。使用新的多重 PCR-RFLP 以及 DNA 测序和 snpBLAST 来检测 MTHFR 677 C>T 和 eNOS +894 G>T 和 eNOS -786 T>C 变体的基因型。使用 Hardy Weinberg 方程计算 MTHFR 677 C>T 和 eNOS +894 G>T 和 eNOS -786 T>C 的等位基因频率。
方法
本研究招募了 114 名健康志愿者，并提取其 DNA。使用 Primer 3 Software version 0.4.0 设计引物对，并针对 BLAST 数据库进行验证。使用单重 PCR 方法测试引物的特异性、功能和退火温度，然后将其组合成单一的多重 PCR。在三个单独的管中进行限制性片段长度多态性（RFLP），然后进行琼脂糖凝胶电泳。纯化 PCR 产物残留物并送去进行 DNA 测序。
结果
MTHFR 677 C>T 的等位基因频率为 0.89（C 等位基因）和 0.11（T 等位基因）；eNOS +894 G>T 的等位基因频率为 0.58（G 等位基因）和 0.43（T 等位基因）；eNOS -786 T>C 的等位基因频率为 0.87（T 等位基因）和 0.13（C 等位基因）。
结论
我们的 PCR-RFLP 方法是一种简单、经济高效且省时的方法。它可以成功地对 MTHFR 677 C>T 和 eNOS +894 G>T 和 eNOS -786 T>C 变体进行基因分型，与 DNA 测序数据的 100%一致性。该方法可常规用于快速研究 MTHFR 677 C>T 和 eNOS +894 G>T 和 eNOS -786 T>C 变体。
Zotero 插件