稳定剂保护PCR扩增dU-DNA效果研究

STABLIZER USED IN PROTECTING dU-DNA PCR PRODUCTS

目的 :了解 94℃灭活UDG与不灭活及扩增产物加稳定剂与不加稳定剂对DNA杂交效率的影响。方法 :采用微孔板DNA杂交技术检测PCR扩增dU -DNA杂交效果。以不加UDG组DNA杂交A值为对照、计算杂交百分率。结果 :不加UDG组扩增前后未经 94℃灭活处理 - 2 0℃保存 2 0h杂交效率为 90 .2 93% ,加UDG组在 4℃、室温、37℃放置 2 0h ,杂交效率为 2 0 .15 % ,2 1.37% ,2 9.37%。加UDG并增加 94℃灭活步骤、37℃水浴放置 2 0h ,杂交效率为 73.5 7% ,78.82 %。在此基础上加入稳定剂 37℃水浴 4 3h杂交效率达到 99.88%～ 10 0 %。 37℃水浴 6 7h杂交效率仍达到 86 .0 6 %～ 97.10 %。结论 :上述结果证实用于抗污染时 ,扩增前后增设 94℃灭活 10min可提高杂交效率。加入稳定剂可使杂交A值 1.80达到对照组A值 1.81水平。提示稳定剂对保护PCR扩增dU -DNA有极其重要作用。

Objective:To investigate whether UDG was inactivated or not and stablizer was added or not effect on hybridization rates.Methods:To apply microwell DNA hybridization technique to detect the hybridization rates of dU-DNA PCR products. When the A(absorbance) value of the group without UDG was acted as control, hybridization rates were worked out. Results:The group without UDG which was not inactivated before and after PCR was stayed at -20℃ for 20 hours ,its hybridization rates was 90.29%, while the hybridization rates of the groups with UDG which stayed at 4℃, room temperature , 37℃ for 20 hours, respectively were 20.15%, 21.37%, 29.37%, respectively. The hybridization rates of the groups with UDG and inactived at 94℃, staying at 37℃ for 20 hours , were 73.57%, 78.82%.On this basis, stablizer was added, staying at 37℃ for 43hours,thus, the hybridization rates ran up to 99.87%～100%. Inspite of the products staying at 37℃ for 67 hours, their hybridization rates were still up to 86.06%～97.10%. Conclusion:In this study, the results of the hybridization rates above showed that during the anti-contamination the hybridization rates could be raised when the products were in activated at 94℃ for 10 minutes. When the stablizer was added ,the A value was 1.80 which came up to the level of the control whose A value was 1.81. The results indicated that stablizer might play a central role at protecting the dU PCR products.