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Department of gene diagnostic method using peputide nucleic acid

肽核酸基因诊断方法系

基本信息

批准号：
09557217
负责人：
UEDA Kunihiro
金额：
$ 7.94万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

我们开发了一种新的诊断方法，使用肽核酸（PNA）。我们首先研究了PNA探针的理化和生物学性质。与DNA探针相比，该探针具有穿透细胞膜的能力强、与靶DNA或RNA的亲和力高、序列特异性强、对核酸酶和蛋白酶有较强的抗性等优点，但其灵敏度仍不足以满足实际应用的需要。然后，我们尝试将联合收割机PNA与CSA（催化信号放大）方法结合，该方法利用与酪胺自由基形成ABC（抗生物素蛋白-生物素复合物）。将该方法应用于MRSA（耐甲氧西林金黄色葡萄球菌）mecA基因的检测，灵敏度明显提高。除了这种PNA-CSA方法，我们研究了其他三种有效的基因诊断方法。第一种是IM（Intercalation-Monitering）-PCR，它能够用荧光探针嵌入双链DNA中实时测量扩增的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基因的点突变。第三种是直接PCR，即不从各种临床样品中分离DNA而进行扩增。我们表明，Ampdirect PCR D1 TM PCR D1是一种商业试剂混合物，可克服内源性抑制物质（如血红蛋白和胆汁酸）对Taq聚合酶的抑制作用，可用于粪便中的直接PCR。