不同固定剂及固定时间对PCR扩增效果的影响

本研究选取五种不同固定液，分别按四种固定时间（12小时，24小时，48小时和72小时）对存在有HBVDNA的人肝癌组织进行固定，脱水包埋。然后提取组织DNA，观察不同固定液及固定时间对PCR扩增效果的影响。同时，还比较了从蜡块中制取PCR模板的四种方法。结果显示：新配中性缓冲甲醛液对组织DNA破坏较小，对PCR扩增效果影响亦较小且成本费用相对较低，固定时间最好不超过48小时。长期放置的用自来水配制的甲醛固定液对组织DNA破坏较大，DNA降解严重，直接影响PCR扩增效果。在用蜡块组织制备DNA模板上，传统酶消化，酚-氯仿抽提，酒精沉淀较为稳定，但所需组织相对较多。对小块组织，充分脱蜡，用双蒸水充分煮沸是制备PCR模板较简便的方法。根据本研究，笔者建议，在目前我国日常病理工作中，应重视对固定液的选择及固定时间的限制，以便能为进一步基因诊断提供先决条件。

The Influence of the Different Fixation Agents and Time on PCR Amplification

Five kuds of fixation agents, according to 4 kinds of different fixation time, were employed to fix hepatic carcinoma tissues in which HBV DNA was presented, then dehydrated and embedded using paraffin.The DNA of the tissues was extracted to investigate the effect of different fixation agents and time on PCR amplification, meanwhile we compared the four different methods by which the template of PCR was prepared from paraffin-embedded tssues. The result showed that the newly prepared neural formalin buffer had minimum damage on DNA, effected lsee on PCR amplification and reduced expenses, but the fixation time should not be more than 48 hours, for tap-water formalin fixation agent damaged DNA seriously, made it degrate seriously, and effected PCR amplification directly. It was a stable method to abstract DNA from paraffin-embedded tssues by using routine enzyme lysing, phenot-chloride extraction and alcohol deposit,but relatively more tissues are needed. It is a easy method to prepare PCR templats by boiling tssues using ddH2O.