Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method ...Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonu-clease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, cir-cularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.展开更多
QuickChange mutagenesis is the method of choice for site-directed mutagenesis （SDM） of target sequences in a plasmid. It can be applied successfully to small plasmids （up to 10 kb）. However, this method cannot eff...QuickChange mutagenesis is the method of choice for site-directed mutagenesis （SDM） of target sequences in a plasmid. It can be applied successfully to small plasmids （up to 10 kb）. However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography （HPLC） purified primers, we were able to achieve SDM in big plasmids （up to 16 kb） involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction （PCR） cycles and 0.5μI of polymerase （instead of 18 PCR cycles and 1.0 μI of enzyme in the standard protocol） were sufficient for the reaction.展开更多
基金Project supported by the Hi-Tech Research and Development (863) Program of China (No. 2007AA02Z151)the National Natural Science Foundation of China (No. 30872223)
文摘Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonu-clease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, cir-cularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.
文摘QuickChange mutagenesis is the method of choice for site-directed mutagenesis （SDM） of target sequences in a plasmid. It can be applied successfully to small plasmids （up to 10 kb）. However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography （HPLC） purified primers, we were able to achieve SDM in big plasmids （up to 16 kb） involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction （PCR） cycles and 0.5μI of polymerase （instead of 18 PCR cycles and 1.0 μI of enzyme in the standard protocol） were sufficient for the reaction.
