Development of tissue fixatives to minimize DNA fragmentation - DNA analysis for formalin-fixed and paraffin-embedded tissues -

开发组织固定剂以尽量减少 DNA 碎片 - 福尔马林固定和石蜡包埋组织的 DNA 分析 -

基本信息

批准号：
09670451
负责人：
TAKAHASHI Masanori
金额：
$ 1.79万
依托单位：
DOKKYO UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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

To minimize degradation of genomic DNA molecules with formalin as the tissue fixative, the additives of EDTA, sodium chloride, methanol and flavonoid were examined.Human tissue specimen was soaked with 10%v/v phosphate-buffered formalin (pH 7.2) and various concentrations of these additives for 24 hours to two years.Thin-sectioned specimen in five μm thick was deparaffinized with xylol and the DNA of five thin-sections in every sample was extracted by means of the phenol-chloroform method.DNA samples extracted were electrophoresed in the agarose gel. Gels were trimmed at 1.2kbp in size.Contents of DNAs extracted from respective gel strips were measured by UV absorbance. Then, these DNAs were amplified by PCR for the loci of D1S80 and some STRs.The results were as follows :For 7 days after fixation, DNAs of more than 1.2kbp decreased drastically to 20% with formalin alone, and to 42% with buffered formalin. EDTA and flavonoid improved 2-4% additively.After the day, no large DNA was remained with formalin alone fixative.Two years later, EDTA addition resulted to be 12% of 1.2kbp DNA content, while other additives were less than 7%. Methanol brought to be worse.Even after 2 years, EDTA fixative gave good template of DNAs of the lung, the kidney and the liver for PCR-based genotyping of D1S80. In addition, TPOX, vWA and THO1 were possibly amplified by more than 500bp size template.EDTA inhibited significantly DNase I activity in the tissues, which concentration was enough at 5mM.Taken all together, EDTA and buffered formalin for tissue fixatives is preferentially recommended.

为了最大程度地减少基因组DNA分子的降解为固定组织，检查了EDTA，氯化钠，甲醇和类黄酮的添加剂。浸泡了10％V/V磷酸盐 - 持续性福尔马蛋白（pH 7.2）的人类组织，以这些添加剂的浓度为24小时，并在24小时内添加了两年的浓度。用二甲醇脱蜡和每个样品中五个薄段的DNA通过苯酚 - 氯仿法提取。将提取的DNA样品电泳在琼脂糖凝胶中电泳。通过大小为1.2kbp修剪凝胶。通过紫外吸光度测量从相对凝胶带中提取的DNA的浓度。然后，通过PCR将这些DNA放大D1S80的局部。结果如下：固定后的7天，单独使用福尔马林的DNA大幅度降低至20％，而使用缓冲福尔马林则为42％。 EDTA和类黄酮又提高了2-4％。一天之后，仅福尔马林固定固定了大的DNA。两年后，EDTA的添加结果占1.2kbp DNA含量的12％，而其他添加剂却小于7％。甲烷醇的情况更糟。此外，TPOX，VWA和THO1可能会被超过500bp的尺寸模板扩增。EDTA在组织中抑制了显着的DNase I活性，在5mm上，浓度在5mm上足够，EDTA和Bufferered hormorin for Thissue for Thissue固定的浓度是固定的。

项目成果

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高橋雅典、加藤幸映、長井敏明、徳留省悟: "DNA解析のためのホルマリン固定法の改良"DNA多型. 7. 67-71 (1999)

Masanori Takahashi、Yukie Kato、Toshiaki Nagai、Shogo Tokudome：“DNA 分析中福尔马林固定方法的改进”DNA 多态性。

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Masanori Takahashi, Hirohisa Nihei, Akira Kurosu, Atuko Saotome, Toshiaki Nagai, Syougo Tokudome: "Effect of EDTA addition to buffered firmaldehyde for DNA degradation-blocking. (in Japanese)"DNA polymorphism. 8. 256-259 (2000)

Masanori Takahashi、Hirohisa Nihei、Akira Kurosu、Atuko Saotome、Toshiaki Nagai、Syougo Tokudome：“缓冲甲醛中添加 EDTA 对 DNA 降解阻断的影响。（日语）”DNA 多态性。

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高橋雅典他: "ヒト組織のホルマリン固定におけるDNA分解抑制のためのEDTA添加の効果"DNA多型. 8. 256-259 (2000)

Masanori Takahashi 等人：“添加 EDTA 对福尔马林固定人体组织过程中 DNA 降解的抑制作用”DNA 多态性。

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Masanori Takahashi, Yukie Kato, Hideaki Kanaya, Akira Kurosu, Toshiaki Nagai and Shigetaro Kamiyama: "Detection of 4 STRs and sex determination using DNA extracted formalin-fixed human tissues by PCR.Advance in research on DNA polymorphisms"Toyoshoten. 13

Masanori Takahashi、Yukie Kato、Hideaki Kanaya、Akira Kurosu、Toshiaki Nagai 和 Shigetaro Kamiyama：“通过 PCR 提取福尔马林固定人体组织中的 DNA 检测 4 STR 和性别确定。DNA 多态性研究进展”Toyoshoten。