应用PCR技术定向引入DNA小片段的策略.pdfVIP

生物技术通报 BI OTECHN OLOG Y B ULLETI N 2005 3 PCR DNA * 1 2 2 2 2 3 张传生 耿立英 孟春花 杨娜娜 朱靖 杜立新 1 2 ( , 271018; , 271018; 3 , 100094)) : 描述一种应用 PCR技术定向引入DNA 小片段和 异酶切位点的方法为了获得 m2/ loxp66EG FPloxp71基因片段根据EGFP基因序列, 设计一对 异引物, 上下游引物分别引入 m2/ loxp66loxp71序列和 Xhol lu1酶切位点以pEGFPN1质粒为模板, 采用 PCR 扩增以合成DNA 双链, 插入到克隆载体 p D18T 对重组子测序结果表明, 实现了DNA 小片段和酶切位点的定向引入 : DNA 重组 DNA 小片段 PCR The Strategy of Directed Introducted Low Molecular Weight DNA by PCR 1 2 2 2 2 3 Zhang Chuansheng Geng Liying eng Chunhua Yang Nana Zhu Jing Du Lixin 1 ( College of Lif e Science , Shandong Agricultural University , Taian 271018; 2 College of A nimal Science and Technology , Shandong A gricultural University , Taian 271018; 3 Animal H usbandry Research Institution of Chinese A gricultural Science College, eij ing 100094) Abstract: A method of directed introduced low DNA fragments and site during construction of recombinant vector was discried .Using pEGFPN1 plasmid as the template ,the gene EGFP was amplified by polymerase chain reaction with a pair of specificcontaining the sites of Xhol, lu1 and these sequences m2/loxp66, loxp71. T hen the m2/ loxp66EGFPloxp71 fragment was subcloned intocloning vector p D18T . Therecombinant plasmid was iden tified by sequencing. The results showed that thesites and low molecular weight DNA weresuccessfully introduced . Key w ords: DNA recombination Low molecular weight DNA PCR DNA , DNA , , ,DNA , , ,DNA R CE( recombinase mediated cassette exchange,), 34bp loxp GFP , DNA , PCR , DNA A , D