尿嘧啶DNA糖苷酶防止PCR产物污染及其对核酸杂交的影响

目的 采用PCR微板核酸杂交ELISA技术分析尿嘧啶DNA糖苷酶（UNG/UDG）防止PCR产物污染及其对PCR扩增效率的影响，探讨在不同A/T含量的微生物中含尿嘧啶的PCR产物对核酸杂交的影响。方法 以3组dUTP含量不同的PCR产物作模板，抗污染组用UNG处理，直接进行再次扩增。用微板核酸杂交ELISA检测第二次PCR扩增的结果。 结果 抗污染组中以含dUTP的PCR产物作模板，结果为阴性；而以不含dUTP的PCR产物作模板，结果为阳性。非抗污染组全部为阳性。对3组PCR产物再进行核酸杂交无明显差别，且在一定温度内，3组具有同样的稳定性。 结论 UNG能有效降解含dUTP的PCR产物，使之不能作为模板再次扩增，可以有效地防止PCR产物的污染。含有尿嘧啶的PCR产物对寡核苷酸探针的杂交无影响。

Prevention of contamination in PCR products by using uracil-DNA-glycosylase before amplification and its influence on nucleic acid hybridization

Objectve To investigate the efficacy of uracil-DNA-glycosylase(UNG) in preventing contamination in the PCR products before amplication procedure and explore its effect on the amplification efficiency. Methods HBV core-region DNA was amplified from HBV genomic plasmid using PCR with or without dUTP. The amplified products were used as the templates for secondary amplification. In anticontamination group, UNG was used to lyse the initially amplified products before secondary amplication in each reaction tube. In contrast, UNG was not used in not-antitamination group. The results of amplification were detected by sandwich micro-well hybridization ELISA technique. Results The templates containing dUTP were not amplified after UNG treatment(negative). In contrast, the templates without dUTP could be amplified(positive). All templates were amplified in not-antitamination group(positive). Conclusion UNG could prevent PCR products contamination effectively, and its effect on amplification efficiency is not visible.