基因扩增技术中抗污染的研究  

Application of Anti-contamination in Gene Amplication Technique

作　　者：韩建德 杜绍财[2] 刘峰[2] 邱国华[2] 

机构地区：[1]北京肝炎试剂研制中心,北京100044 [2]北京大学人民医院肝病研究所

出　　处：《中国医师杂志》2004年第12期1594-1596,共3页Journal of Chinese Physician

基　　金：卫生部"九五"攻关资助项目 (96 - 90 6 - 0 3 - 0 6)

摘　　要：目的　了解残留UDG对dU -DNAPCR产物的影响及灭活UDG时对Taq -P的影响。方法　采用微孔板杂交技术检测dU -DNAPCR产物 3 7℃放置 2 0h杂交百分率 ;检测在 -2 0℃、4℃、室温、3 7℃时不同保存条件下的杂交百分率 ;94℃灭活 10min对Taq -P活性的影响。结果　加 0 2单位、1 0单位UDG组比不加UDG组杂交A值下降 13 2 81%～ 2 0 5 5 7%。t检验差异有显著性 (t=2 85 5 ,2 869,P <0 0 5 ) ;经 94℃灭活产物组 3 7℃杂交A值比 -2 0℃A值下降 5 5 47% ,未经 94℃灭活杂交A值下降 10 3 45 % ;94℃预变性 3 0s杂交A值为 98 714 %、10min 3 0s组杂交A值为 96 818%。结论　以上结果提示经 94℃加热灭活对PCR产物保存有重要作用 ,不仅对灭活UDG有作用而且还可灭活来源于UDG以外可破坏DNA的酶。表明含UDG组杂交A值比不加UDG组低13 2 81%～ 2 0 5 5 7%与UDG有关。Objective To explore the effect of residual UDG on all dU-DNA PCR products. Methods The hybridization percentage of dU-DNA PCR products, which were stored at 37℃ for 20 hours as well as at -20℃,4℃,room temperature and 37℃,respectively, were detected by employing microplate hybridization technique. The effect of inactivating the products at 94 ℃ for 10 minutes on the activity of Taq polymerase was also analysed. Results Compared with the control, A value decreased by 13.281% and 20.557%, respectively after adding 0.2u and 10u UDG (P<0.01). A value of the products inactivated at 94 ℃ and hybridized at 37℃ decreased by 5.54% compared with hybridized at -20℃, and A value of the products not inactivated at 94 ℃ decreased by 10.345%. A value was 98.714% when the products were predenatured at 94 ℃ for 30 seconds, and 96.818% for 10 minutes. Conclusion The results indicated that the inactivation of PCR products at 94 ℃ played an important role in the conservation of PCR products through inactivating UDG and the other enzymes from different sources.

关 键 词：灭活 术中 基因扩增技术 活产 微孔板杂交技术 DNA 放置 UDC 百分率 结论 

分 类 号：R346[医药卫生—基础医学]