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同源引物的非等量PCR 被引量:3

Homologous Primer Unequal PCR
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摘要 为确定引物量对同源引物扩增DNA的影响.将正向引物GFO与同源度为45%、90%、100%、54%、9%的反向引物CR1、CR2、CR3、CR4、CR5分别用于扩增5.9 kb p ET20b-C2-G10-C2模式DNA.等量引物(500 nmol/L)时,CR3和CR4均无法扩增目的条带.而非等量PCR时,将反向引物量降低10倍(50 nmol/L),CR3和CR4均扩增出目的条带,即正向引物量降低10倍时,五对引物均扩增出目的条带.CR3降低100倍、CR4降低10倍扩增DNA最多,经15~20次循环五对引物扩增DNA最多.实验结果表明,非等量PCR通过降低单侧引物量减少引物二聚体形成,从而扩增双链DNA. To analyze the quantity effect of homologous primers on DNA amplification,forward primer GFO having 45%,90%,100%,64%,9%complementation with reverse primer CR1,CR2,CR3,CR4,CR5 were designed to amplify a 5.9 kb model linearized plasmid DNA pET20b-C2-G10-C2 containing an Aspergillus niger GH10 xylanase gene.Compared with non-producing target DNAs by equal quantities of the primers CR3 and CR4 with the GFO,the target DNA bands were amplified by 10-fold decreased primers CR3 and CR4.All target DNA bands were amplified by the five primers with 10-fold decreased primer GFO,verifying that equal quantities of homologous primers inhibited DNA amplifications.Maximal DNAs were amplified with the 100-fold decreased CR3 and the 10-fold decreased CR4.Maximal DNAs of 1.4-2.2μg were amplified by the decreased quantities five homologous primers after 15-20 cycles,similar to that of normal PCR amplification.Different from single-strand DNA amplification of asymmetric non-homologous primer PCR,the unequal quantity primer PCR amplified doublestrand DNAs by decreasing complementation effect of homologous primers.
作者 上官云杰 梁亚萍 杨昂 程晋生 黄亚威 刘亮伟 SHANGGUAN Yunjie;LIANG Yaping;YANG Ang;CHENG Jinsheng;HUANG Yawei;LIU Liangwei(Life Science College,Henan Agricultural University,Zhengzhou 450002,China;Key Laboratory of Enzyme Engineering of Agricultural Microbiology,Zhengzhou 450002,China)
出处 《河南科学》 2018年第3期326-333,共8页 Henan Science
基金 国家自然科学基金项目(31371831 31771915)
关键词 非等量 同源引物 PCR DNA unequal quantity complementary primer PCR DNA
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