Ichikawa Y, Ishikawa T, Momiyama N, Chishima T, Hasegawa S, Yamaoka H, Yamamoto N, Okamoto T, Kawaguchi M, Shimada H
Second Department of Surgery, Yokohama City University School of Medicine, Japan.
Scand J Clin Lab Invest. 1996 Nov;56(7):641-7. doi: 10.3109/00365519609090599.
Polymerase chain reaction (PCR) is a powerful tool for clinical diagnosis in the field of oncology, infection, and allergy. However, a simple and sensitive method for quantifying PCR products has not been established. We therefore used a novel fluorescence DNA intercalator, pyrylium iodide (P2) to quantify PCR products. A 838-bp fragment of the human beta-actin gene and a 374-bp fragment of the gene for ornithine decarboxylase (ODC) and ODC primer dimers were generated by PCR and quantified using either P2 or another fluorescence DNA intercalator, YOYO-1. In addition, utilizing RT-PCR and the P2 method, the ODC mRNA expression from 10 colonic cancers was quantified. Serially diluted beta-actin and ODC PCR products could be quantified using P2 without having to separate them from primer dimers and other components of the reaction mixture. However, YOYO-1 could not be used to quantify the PCR products because of high background fluorescence from the primer dimers. In the clinical study, ODC mRNA expression as quantified by P2 was significantly higher in cancerous tissue (113.8 +/- 4.1; mean +/- SEM; plate reader units) than in mucosa of normal appearance (68.4 +/- 4.8). P2 is a promising tool for quantifying PCR products.
聚合酶链反应（PCR）是肿瘤学、感染和过敏领域临床诊断的有力工具。然而，尚未建立一种简单且灵敏的定量PCR产物的方法。因此，我们使用了一种新型荧光DNA嵌入剂碘化吡喃鎓（P2）来定量PCR产物。通过PCR产生人β-肌动蛋白基因的838bp片段、鸟氨酸脱羧酶（ODC）基因的374bp片段以及ODC引物二聚体，并使用P2或另一种荧光DNA嵌入剂YOYO-1进行定量。此外，利用逆转录PCR和P2方法，对10例结肠癌的ODC mRNA表达进行了定量。连续稀释的β-肌动蛋白和ODC PCR产物可以使用P2进行定量，而无需将它们与引物二聚体及反应混合物的其他成分分离。然而，由于引物二聚体产生的高背景荧光，YOYO-1不能用于定量PCR产物。在临床研究中，用P2定量的癌组织中ODC mRNA表达（113.8±4.1；平均值±标准误；酶标仪单位）显著高于外观正常的黏膜组织（68.4±4.8）。P2是一种很有前景的定量PCR产物的工具。
