粘质沙雷氏菌
核酸酶
聚合酶链反应
生物
热启动PCR
分子生物学
大肠杆菌
DNA
基因
微生物学
化学
多重聚合酶链反应
生物化学
作者
Jiaxin Cai,Yunyi Zhang,Xiaoyue Jiang,Yaohua Hu,Wei Chen
摘要
Abstract Polymerase chain reaction (PCR) is an important tool for exogenous gene acquisition and recombinants identification. There exist two problems when using Serratia marcescens as a template for PCR amplification: amplified PCR products are rapidly degraded, and the results of PCR amplification are unstable. The aim of the present work was to elucidate the reasons for this. By mixing PCR products amplified from Escherichia coli DH5α with S. marcescens supernatant or pellet, we found that the DNA‐degrading substance in S. marcescens is thermally resistant and present both intracellularly and extracellularly. We then determined that it is protein, and most likely S. marcescens nuclease, that degrades PCR products since the addition of SDS and EDTA can effectively inhibit or block the degradation of PCR products. By knocking out the S. marcescens nuclease encoding gene, nucA , we confirmed that the nuclease is responsible for the degradation of PCR products and the instability of PCR amplification. This work is the first to show that the S. marcescens nuclease is temporarily and partially inhibited by high temperatures during PCR and recovers rapidly at room temperature after PCR.
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