Improvement of DNA extracting method from formalin-fixed paraffin-embedded tissue

福尔马林固定石蜡包埋组织DNA提取方法的改进

• 批准号: 10670186
• 负责人: MUKAI Kiyoshi
• 金额: $ 1.66万
• 依托单位: Tokyo Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670186/
• 关键词: Pathology; PCR; DNA; SSCP; Formalin; Paraffin; ホルマリン固定; パラフィンブロック; 病理組織標本

It is widely used to extract DNA from formalin-fixed, paraffin-embedded pathology material and use it for genetic analysis by PCR method, However, DNA in paraffin-embedded tissue is modified and degraded during fixation/embedding processes and it is difficult to constantly obtain adequate amount and quality of DNA. Therefore, DNA from fixed tissue cannot be used for Southern blot analysis and the results of PCR analysis are inconsistent. It is the purpose of this study to devise an improved DNA extraction method that can obtain larger amount of good quality DNA from fixed and embedded tissue than the conventional method using phenol/chloroform extraction. Conventional method of phenol/choroloform extraction after xylene deparaffinization and proteinase K digestion was used as a control for the amount and quality of extracted DNA. First, the temperature of deparaffinization or proteinase K digestion was raised. Another method applied microwave irradiation before proteinase K digestion. The last method used heating by microwave irradiation for deparaffinization. The last method consistently extracted more DNA and efficiency of PCR was higher than the other methods. DNA extracted by this method was suitable for PCR-SSCP analysis or direct sequencing of nucleic acids.

广泛用于从福尔马林固定、石蜡包埋的病理材料中提取DNA，并用PCR方法进行遗传分析，但石蜡包埋组织中的DNA在固定/包埋过程中会发生修饰和降解，难以持续稳定地检测。获得足够数量和质量的 DNA。因此，来自固定组织的DNA不能用于Southern印迹分析并且PCR分析的结果不一致。本研究的目的是设计一种改进的 DNA 提取方法，与使用苯酚/氯仿提取的传统方法相比，该方法可以从固定和包埋的组织中获得大量优质 DNA。采用二甲苯脱蜡和蛋白酶 K 消化后苯酚/氯仿提取的常规方法作为提取 DNA 的数量和质量的对照。首先，提高脱蜡或蛋白酶K消化的温度。另一种方法是在蛋白酶 K 消化之前应用微波照射。最后一种方法是通过微波照射加热进行脱蜡。最后一种方法始终提取更多的 DNA，并且 PCR 效率高于其他方法。该方法提取的DNA适用于PCR-SSCP分析或核酸直接测序。

项目成果

杉江利佳子、佐藤雄一、向井清: "ホルマリン固定・パラフィン包理組織からのDNA抽出法の検討"投稿準備中.

Rikako Sugie、Yuichi Sato、Kiyoshi Mukai：“从福尔马林固定和石蜡包埋的组织中提取 DNA 方法的研究”，准备提交。