Institute for Agro-food Standards and Testing Technology, Laboratory of Quality & Safety Risk Assessment for Agro-products (Shanghai), Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China.
Department of Chemistry and Biochemistry, Florida International University, 11200 SW Eighth Street, Miami, Florida, 33199, United States.
Sci Rep. 2018 Oct 5;8(1):14858. doi: 10.1038/s41598-018-33223-2.
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.
各种添加剂可以提高 PCR 扩增的质量，但这些通常需要相当多的优化才能达到最佳性能。在这里，我们证明了使用巯基修饰的引物可以提高 PCR 的灵敏度和产量。在对副溶血性弧菌基因组 DNA 的实验中，这种引物修饰将 PCR 灵敏度提高了 100 多倍，同时提高了扩增子的产量。然后，设计了一个具有相同引物结合区域和不同内部扩增序列的人工质粒。结果表明，使用相同的巯基修饰引物也可以提高扩增。这表明增强不是由于硫醇修饰的引物对扩增序列的二级结构的影响。随后的实验表明，这种修饰的效果可能是由于引物与反应混合物中的蛋白质之间的相互作用发生了改变。与标准 DNA 引物相比，带有巯基修饰的引物的扩增受到外来蛋白质的强烈抑制，这表明由于与这些蛋白质的相互作用，巯基修饰的引物可能受到抑制。然而，在无污染物的反应中，巯基修饰的引物可能与 DNA 聚合酶更强烈地相互作用，从而可以改善 PCR 扩增。
