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聚合酶链反应(PCR)灵敏度极高,痕量的PCR产物能污染新的PCR体系(称为产物污染)导致假阳性结果,我们通过在所有的PCR扩增中掺入dUTP使PCR产物含“dU”,在以后的PCR扩增前用尿嘧啶-DNA糖基化酶(Uracil-DNA-Glycolase,UDG)处理,然后加热去除UDG活性防止了产物污染。UDG切割磷酸糖骨架上的尿嘧啶,可阴止DNA聚合酶对它的复制,而对普通DNA(即含”dT”)无影响。因为UDG不与dUTP反应,而且在PCR扩增前加热变性失活,使含尿嘧啶的污染物的污染得到很好控制。
Polymerase chain reaction (PCR) sensitivity is extremely high. Trace amounts of PCR products can contaminate the new PCR system (called product contamination) resulting in false-positive results. We have incorporated PCR products with “dUTP” dU “and treated with Uracil-DNA-Glycolase (UDG) prior to subsequent PCR amplification, followed by heating to remove UDG activity and prevent product contamination. UDG cleaves uracil on the sugar-sugar backbone and repeats its replication by DNA polymerase, with no effect on normal DNA (ie containing ”dT"). Because UDG does not react with dUTP, and heat denaturation inactivates before PCR amplification, contaminating uracil-containing contaminants is well controlled.