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目的 :重叠聚合酶链反应 (OverlappingPolymeraseChainReactionPCR)法是在上下游引物之间设计一对碱基互补的嵌套引物 ,第一轮PCR分别扩增 5’端和 3’端片段 ,第二轮PCR以第一轮PCR的 5’端和 3’端片段混和物为模板 ,用上下游引物扩增出全长片段。该文用逆转录 -OverlappingPCR法快速高效地从前骨髓干细胞中扩增到人类PU .1全长cDNA为 44 2bp ,酶切和测序证实完全与前人报道一致。构建的真核表达质粒pcDNA3.1-hPU .1可以被特异的抗体识别 ,体外细胞株转染证实具有特异的激活启动子活性的功能。
Objective: Overlapping Polymerase Chain Reaction (PCR) method is to design a pair of base-complementary nested primers between upstream and downstream primers. The first round of PCR amplifies the 5 ’end and the 3’ end respectively. The second round of PCR In the first round of PCR, the mixture of 5 ’end and 3’ end fragments was used as a template, and the full length fragment was amplified by using the upstream primer and the downstream primer. In this study, we rapidly and efficiently amplified human PU from pre-bone marrow stem cells by Reverse-transcription-OverlappingPCR method.1 The full-length cDNA was 44 2bp, and confirmed by restriction enzyme digestion and sequencing. The constructed eukaryotic expression plasmid pcDNA3.1-hPU.1 can be recognized by specific antibodies. Transfection of in vitro cell lines confirmed the function of activating specific promoter.