Detection of catechol-O-methyltransferase Val158Met polymorphism by a simple one-step tetra-primer amplification refractory mutation system-PCR
Detection of catechol-O-methyltransferase Val158Met polymorphism by a simple one-step tetra-primer amplification refractory mutation system-PCR
复制标题
DOI：
10.1016/j.mcp.2006.12.001
复制
发表时间：
2007-06-01
影响因子：
3.3
通讯作者：
Ruiz-Larrea, Maria Begona
中科院分区：
文献类型：
--
作者：
Ruiz-Sanz, Jose Ignacio;Aurrekoetxea, Igor;Ruiz-Larrea, Maria Begona
人儿茶酚-O-甲基转移酶（COM7 基因）第 21881 号核苷酸处的 G -> A 转变代表一种功能性遗传多态性 (Va1158Met)，使酶活性降低，与精神疾病和雌激素相关癌症相关。描述了一种基于四引物检测这种多态性的新方法 扩增难治性突变系统聚合酶链式反应 (ARMS-PCR)，使用单个 PCR 来区分两个等位基因。两个引物扩增一个共同的扩增子，与所考虑的等位基因无关。同时使用两个引物，其3'碱基不同。在 Val/Val 或 Met/Met 条件下，一般扩增子和特定等位基因均发生扩增；在 Val/Met 条件下三种不同 产生扩增子。对含有 G/A 多态性的 COMT 区域进行直接 DNA 测序证明了这种四引物 ARMS-PCR 方法的有效性。 PCR-RFLP 重新评估显示基因型归属 100% 一致。西班牙人群中携带 COMTHH 基因型的受试者占 28%，COMLL 纯合子占 21%。所描述的方法提供了一种快速且可靠的 确定 COMT 多态性的方法可用于使用最少量 DNA 的大型临床研究，避免及时且昂贵地使用限制性酶。 (c) 2007 Elsevier Ltd. 保留所有权利。
The G -> A transition at nucleotide 21881 of the human catechol-O-niethyltramsferase (COM7 gene represents a functional genetic polymorphism (Va1158Met), rendering an enzyme with reduced activity that has been associated with psychiatric disorders and estrogen related cancers. A new method for the detection of this polymorphism is described, based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), with a single PCR to discriminate both alleles. Two primers amplify a common amplicon independently of the allele considered. At the same time, two primers are used, differing in the 3' base. In the Val/Val or Met/Met conditions, amplification occurs both in the general amplicon and in the specific allele; in the Val/Met condition three different amplicons are produced. Direct DNA sequencing of a COMT region containing the G/A polymorphism demonstrates the validity of this tetra-primer ARMS-PCR method. Reevaluation by PCR-RFLP revealed 100% accordance for genotype adscription. Subjects carrying the COMTHH genotype in a Spanish population comprised 28%, and the COMLL homozygotes amounted to 21%. The described method provides a fast and reliable approach for determining COMT polymorphism that can be useful in large clinical studies using minimal quantity of DNA, avoiding the timely and costly use of restriction enzymes. (c) 2007 Elsevier Ltd. All rights reserved.