Yagi N, Satonaka K, Horio M, Shimogaki H, Tokuda Y, Maeda S
Department of Pathology, Kobe University School of Medicine, Japan.
Biotech Histochem. 1996 May;71(3):123-9. doi: 10.3109/10520299609117148.
Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.
本文介绍了从适合基因分析的甲醛固定组织中降解和提取高分子量DNA的方法。我们之前报道过，DNA酶可能在从甲醛固定组织中提取的DNA降解过程中起重要作用（Tokuda等人，1990年）。在本研究中，室温下用缓冲甲醛固定的大鼠组织上清液中的DNA酶活性在3小时内可忽略不计。对从重组染色质中提取的DNA进行分析发现，在没有DNA酶的情况下，降解程度会随着甲醛固定时间的延长而增加。此外，从固定在含有EDTA的缓冲甲醛中的无DNA酶活性的组织中可以提取高分子量DNA。这些结果表明，DNA降解主要是由一种不同于DNA酶的机制引起的，而这种机制会被EDTA抑制。对于临床应用，通过Southern印迹法成功地从特别是在4℃下用缓冲甲醛固定的大鼠脾脏组织中检测到了v-H-ras基因。低温固定对于基因分析很有用。
