采用十二烷基硫酸钠抑制PCR产物中残留尿苷酶

Stablizer used in inhibiting residual uracil DNA glycosylase activity in the PCR products

目的 :研究抑制PCR产物中残留尿苷酶 (uracilDNAglycosylase ,UDG)活性的试剂。 方法 :采用DNA EIA技术检测在全dU DNA扩增物中含有稳定剂和不含稳定剂的样品 ,置室温 2 4h和 37℃ 4 8h的DNA杂交百分率。结果 :(1)在PCR产物中加入稳定剂A液和B液置室温 2 4h ,取 5 μl直接杂交 ,其杂交百分率为 6 1.4 70 %和6 8.996 % ,对照组为 99.2 83%。 (2 )在 30 μlPCR产物中加入稳定剂后 ,并加 1×PCR缓冲液稀释至 70 μl,置 37℃4 8h取 10 μl样品杂交 ,5 μl和 2 .5 μlA液组杂交百分率为 71.0 92 %和 6 7.6 91% ,对照组为 6 8.0 2 0 %和 6 3.90 6 %。5 μl和 2 .5 μlB液组杂交百分率为 91.333%和 80 .30 7% ,对照组为 6 3.90 6 %和 6 8.0 2 0 %。 结论 :B液可抑制PCR产物残留UDG活性 ,对保护全dU

Objective: To research and make the agent for inhibiting activity of residual UDG in PCR products. Methods:DNA EIA technique was applied to detect the hybridization rates of all dU DNA PCR products containing stablizer and without stablizer, the hybridization rates at room temperature for 24 hours and at 37 ℃ for 48 hours. Results: (1)Stablizers A and B were added in the PCR products of which 5 μl was taken out for hybridization and their hybridization rates were 61.470%, 68.996% ,respectively. The hybridization rate of the control was 99.283%.(2) After 30 μl of the PCR products were added to stablizers, diluted to 70 μl with 1×PCR buffer ,and then kept at 37 ℃ for 48 hours 10μl of which was taken out for hybridization. Hybridization rates with 5 μl and 2.5 μl of stablizers A were 71.092% and 67.691%,respectively. The controls were 68.020% and 63.906%.The hybridization rates of the groups with stablizer B were 91.333% and 80.307%,The controls were 63.906% and 68.020%. Conclusion: In this study, the results of the hybridization rates with DNA EIA method revealed that hybridization of the PCR products which were not diluted and were directly added to the stablizer was inhibited .In contrast, hybridization of the PCR products added to the and diluted was not inhibited. After the mixture of PCR products and the stablizer were diluted, hybridization rates with stablizer A increased by 3.072% and 3.095% as compared with that of controls. Accordingly, hybridization rates of the groups with stablizer B increased by 27.427% and 12.287% as compared with that of controls. The results above indicate that stablizer B may inhibit the activity of residual UDG and play a central role in protecting all dU DNA PCR products.