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本文采用基于ＳＶ４０病毒的新型穿梭质粒ｐＳＰ１８９，作为ＰＺ１８９的衍生质粒其独特性在于ｐＳＰ１８９是一个质粒群（ＰｌａｓｍｉｄＰｏｐｕｌａｔｉｏｎ）每个质粒在其靶基因ｓｕｐＦＴＲＮＡ的３’端都插入一段随机产生的８－ｂｐ的标识序列ＳｉｇｎａｔｕｒｅＳｅｑｕｅｎｃｅ），通过对突变子靶基因ＳｕｐＦ和标识序列同时测序可以鉴别出一个转染细胞瓶中两个靶基因序列改变相同的突变子是独立突变（ＩｎｄｅｐｅｎｄｅｎｔＭｕｔａｔｉｏｎ）还是同胞突变（ＳｉｂｌｉｎｇＭｕｔａｔｉｏｎ）。由于ｐＳＰ１８９的独特性，我们总结出一套简便、快速的质粒ＤＮＡ转化、扩增保存、提取、转染及从哺乳动物细胞中回收纯化的方法，结果表明，用我们的一步法从细菌中提质粒和改良的Ｈｉｒｔ法从细胞中回收质粒基本没有细菌和细胞ＤＮＡ的污染，而且不需超离心即可达到较高的纯度并获得较高的转化率和转染率，为进一步筛选分离突变体并对靶基因进行ＤＮＡ序列分析打下了基础。
In this paper, a novel shuttle plasmid pSP189 based on SV40 virus was used as a derivative plasmid of PZ189. The uniqueness of this plasmid is that pSP189 is a plasmid group. Each plasmid inserts a randomly generated 8-bp SignatureSequence). By sequencing the SupF and Marker sequences of the mutant target genes simultaneously, it is possible to identify whether Mutation Mutations (Mutations Mutated Mutations or Sibling Mutations) of two target gene sequences in a transfection flask are the same. Because of the unique nature of pSP189, we conclude with a simple and rapid plasmid DNA transformation, amplification, preservation, extraction, transfection and purification method from mammalian cells. The results show that using our one-step method from bacteria Plasmids and Modified Hirt Methods The recovery of plasmids from cells is virtually free of bacterial and cellular DNA contamination and achieves higher purity and higher conversion and transfection rates without ultracentrifugation for further screening of mutants And the target gene DNA sequence analysis laid the foundation.