石蜡包埋组织提取DNA不同条件下的PCR扩增

背景:在甲醛固定石蜡包埋组织的制作及保存过程中会对DNA造成损害,提取的DNA在用于PCR扩增时部分结果并不理想。目的:比较分析石蜡包埋组织中提取DNA应用于PCR反应的影响因素。方法:从10.0μm×2、10.0μm×4和10.0μm×5石蜡包埋食管癌组织切片中提取DNA,以0.05,0.1,0.2μg模板DNA量扩增内参基因β-actin,PCR反应循环次数分别为35,40,45次,分析影响PCR反应的因素。结果与结论:琼脂糖凝胶电泳结果显示以10.0μm×2组织切片量,PCR反应循环次数40次,PCR反应模板DNA量0.05μg扩增取得的目的基因DNA的质量最高。说明减少石蜡包埋组织用量和减少模板DNA量有助于获得高质量的PCR反应条带,且PCR反应循环次数应大于40次,小于45次,再增加循环次数,则意义不大。

PCR amplication of the DNA extracted from paraffin-embedded tissues in different conditions

BACKGROUND: Because of the damage of formalin-fixed paraffin-embedded tissue to DNA in the process of its production and preservation, it is difficult to extract high quality and sufficient DNA for PCR. OBJECTIVE: To analyze the influencing factors of the DNA extracted from paraffin-embedded tissue for PCR. METHODS: Genomic DNA was extracted from paraffin-embedded esophageal carcinoma tissue sections of 10.0 μm×2, 10.0 μm×4 and 10.0 μm×5. Template DNA amount of 0.05, 0.1, 0.2 μg was used to amplify gene β-actin, respectively. PCR cycle times were set as 35, 40 and 45. The influencing factors of PCR were analyzed. RESULTS AND CONCLUSION: The analysis of agarose gel electrophoresis showed that when paraffin-embedded esophageal carcinoma tissue section amount was 10.0 μm×2, the PCR cycle times were 40; when the template DNA amount was 0.05μg, the obtained target DNA was of the highest quality. It shows that reducing the amount of paraffin-embedded tissue and DNA template can be help to require high quality PCR strips; PCR cycle times should be more than 40 and less than 45, and if it increases beyond this range there is meaningless.