摘要
目的制备含鸭乙型肝炎病毒（ＤＨＢＶ）前核心区终止密码突变的基因片段，以对此基因突变作有关生物学研究。方法用重叠延伸聚合酶链反应（ＯＥ－ＰＣＲ）制备含此人工定向点突变的基因片段；用不对称聚合酶链反应（ａｓｙ－ＰＣＲ）制备单链ＤＮＡ模板作测序。结果凝胶电泳显示ＯＥ－ＰＣＲ产物与克隆ＤＨＢＶＤＮＡ酶切片段位置一致，测序证实该点突变存在。结论用ＯＥ－ＰＣＲ作人工定向基因突变，不需要克隆制备单链模板及筛选阳性克隆，２天内即可出结果，较传统方法简便、快速、有效。
ObjectiveTogenerateaDNAfragmentcontainingastopcodonmutationinprecoreregionofduckhepatitisBvirusforbiologicalresearch.MethodsThispointmutationoftheDNAfragmentwascreatedbyoverlapextensionusingpolymerasechainreaction(PCR).ThesinglestrandDNAtemplatewaspreparedbyasymmetricPCRforDNAsequencing.ResultsAfterdigestionwithNco1andNsi1thebandofPCRproductsranthesamepositionthroughgelelectrophoresewithclonedDHBV3fragment.ThepointmutationwasconfirmedbydirectedDNAsequencing.Conclu-sionsEliminationofclonestepforsinglestrandDNAtemplateandscreeningpositiveclonesandtheadvantagesofsite-directedmutagenesisbyoverlapextensionusingPCRareitssimplicityandeficien-cy.Theprocedurecanbeperformedwhthintwodays.
