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Enhancement of PCR Sensitivity and Yield Using Thiol-modified Primers

文献类型： 外文期刊

作者： Bai, Yalong 1 ; Xiao, Yi 2 ; Suo, Yujuan 1 ; Shen, Yuanyuan 1 ; Shao, Yi 1 ; Zhang, Donglai 1 ; Zhou, Changyan 1 ;

作者机构： 1.Shanghai Acad Agr Sci, Minist Agr, Lab Qual & Safety Risk Assessment Agroprod Shangh, Inst Agrofood Stand & Testing Technol, Shanghai 201403, Peoples R China

2.Florida Int Univ, Dept Chem & Biochem, 11200 SW Eighth St, Miami, FL 3

期刊名称：SCIENTIFIC REPORTS （ 影响因子：4.379； 五年影响因子：5.133 ）

ISSN： 2045-2322

年卷期： 2018 年 8 卷

页码：

收录情况： SCI

摘要： Various additives can enhance the quality of PCR amplification, but these generally require considerable optimization to achieve peak performance. Here, we demonstrate that the use of thiol-modified primers can enhance both PCR sensitivity and yield. In experiments with V. parahaemolyticus genomic DNA, this primer modification enhances PCR sensitivity by more than 100-fold, with accompanying improvements in amplicon yield. Then, an artificial plasmid with the same primer binding regions and different internal amplification sequence was designed. The result showed that the amplification also be improved by using the same thiol-modified primers. It indicated the enhancement was not caused by the effect of the thiol-modified primers on the second structure of amplification sequence. Subsequent experiments demonstrate that the effects of this modification are potentially due to altered interaction between the primers and proteins in the reaction mixture. Amplification with thiol-modified primers was strongly inhibited by the presence of extraneous proteins relative to standard DNA primers, which indicates that thiol-modified primers may be inhibited due to interaction with these proteins. In contaminant-free reactions, however, the thiol-modified primers might interact more strongly with DNA polymerase, which could in turn improve PCR amplification.

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