通过PCR进行长片段DNA的合成  

Use PCR Synthesis Large Fragment DNA

作　　者：李炜东[1] 梁布锋[1] 祁自柏[1] 

机构地区：[1]中国科学院武汉病毒研究所,武汉430071

出　　处：《遗传》2004年第3期349-352,共4页Hereditas（Beijing)

基　　金：北京市科委基金(项目编号H030230001130)~~

摘　　要：利用PCR合成DNA长片段(Synthesis Large Frament DNA using PCR,SLFD PCR)是一种有效的合成长片段DNA的方法。采用一段已知的500～600bp碱基的DNA片段为PCR模板,根据所要合成的DNA序列可以设计一系列的PCR引物,这些引物都位于模板DNA的5’端,长度为50～60bp,且从5’到3’方向顺序重叠,重叠碱基数目为12～15,全部引物叠加所得到的DNA正是自己所要合成的DNA。这组引物中最3’端的一条含有一个BamH Ⅰ酶切位点,在该位点后面有15碱基与模板DNA5’端一致的序列。另外还设计一条与该模板匹配的下游引物,引物内也含有一个BamH Ⅰ酶切位点。首先采用5’端最右侧的引物与下游引物进行PCR,在PCR进行10个循环后,以此次PCR的产物为下一轮PCR的模板,该轮PCR采用右侧倒数第二个引物为上游引物,下游引物保持不变。采用类似的方法,完成所有的PCR循环,就可以得到所需要合成的DNA长片段。该方法尤其适合100～200碱基左右的长片段DNA的快速合成与克隆。Synthesis Large Frament DNA using PCR (SLFD PCR) is a useful method to synthesis large fragment DNA. Use a knowed 500 ～ 600 basepair DNA fragment as PCR templet, a series of 5' terminal primers are de- signed, and these primers are overlap one by one from 5' terminal to 3' terminal, the net DNA is just what you want to synthesis. The last 3' terminal primer of these primers has a BamH I site, and behind the BamH I site there are 15 bp overlap the 5' terminal of the templet. Another downstream primer complement the 3' terminal of the tem- plet, and has a BamH I site too. PCR begin using the innerest 5' terminal primer and the downstream primer. After 10 cycles of PCR, use the product of the PCR as the templet of next round PCR, but this time the upstream primer change to the 5' terminal outer primer. So do the PCRs, till all the 5' terminal primers take part in the PCR. Clone the final PCR product and BamH I cut the original DNA templet, The DNA synthesis complete. It's a usefull meth- od to synthesis 100～200bp DNA fragment, even more long DNA fragment.

关 键 词：PCR DNA合成 长片段 

分 类 号：Q781[生物学—分子生物学]