Department of gene diagnostic method using peputide nucleic acid

我们开发了一种使用 PNA（肽核酸）的新型诊断方法。我们首先检查了 PNA 探针的物理化学和生物学特性。我们确认了其相对于DNA探针的优势，例如能够自由穿透细胞膜、对DNA或RNA靶标的高亲和力、精确的序列特异性以及对核酸酶和蛋白酶的抗性，但其灵敏度被证明不足以实际使用。然后，我们尝试将 PNA 与 CSA（催化信号放大）方法结合起来，该方法利用 ABC（亲和素-生物素复合物）与酪胺自由基形成。我们将该组合应用于MRSA（耐甲氧西林金黄色葡萄球菌）mecA基因的检测，并获得了灵敏度的显着提高。除了这种 PNA-CSA 方法之外，我们还研究了其他三种有效基因诊断的方法。第一种是 IM（嵌入监测）-PCR，它能够使用插入双链 DNA 的荧光探针实时测量扩增的 DNA。第二种是原位 PCR，一种扩增病理标本中 DNA 的技术。我们的方法能够检测肺癌组织中 K-ras 基因酸的点突变。第三种是直接PCR，即无需从各种临床样本中分离DNA即可进行扩增。我们证明，Ampdirect 是一种商业试剂混合物，可克服血红蛋白和胆汁等内源性抑制物质对 Taq 聚合酶的抑制，可用于直接 PCR 以及粪便。

We developed a novel diagnostic method using PNA (Peptide Nucleic Acid). We first examined physicochemical and biological properties of PNA probe. We confirmed its characters superiority over DNA probe, such as the ability to freely penetrate through cell membranes, a high affinity to DNA or RNA target, the precise sequence specificity, and resistances to nuclease and protease, but the sensitivity proved to be insufficient for practical use. We then tried to combine PNA with the CSA (Catalyzed Signal Amplification) method, which utilizes ABC (avidin-biotin complex) formation with tyramine radicals. We applied this combination to detection of mecA gene in MRSA (methicillin-resistant Staphylococcus aureus), and obtained a remarkable improvement of the sensitivity. In addition to this PNA-CSA method, we investigated three other methods for efficient gene diagnosis. The first is IM (Intercalation-Monitering)-PCR, which enabled a real-time measurement of amplified DNA with a fluorescent probe to be intercalated into double-strand DNA. The second is in situ PCR, a technique to amplify DNA in pathological specimen. Our method enabled detection of point mutation of K-ras gene in lung cancer tissues. The third is direct PCR, that is amplification without DNA isolation from various clinical samples. We showed that AmpdirectィイD1TMィエD1, a commercial reagent cocktail to overcome inhibition of Taq polymerase by endogenous inhibitory substances, such as hemoglobin and bile acids, is useful for direct PCR from as well as feces.