[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension...[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.展开更多
[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlappe...[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlapped and served as the template to the other DNA strand in amplification. Then, several rounds of over- lap PCR reaction were conducted to mutate the four cysteines in the HP intein into serines, and introduce a linker sequence containing a tag for product detection and purification. [Result] DNA sequencing analysis proved that the four cysteines in the HP intein were successfully mutated into serines; the PCR precursor fragments were also rejoined into an intact HP gene fragment, without introducing any other unex- pected mutation; the DNA linker of 46 bp was successfully inserted into the de- signed HP gene sequences, without any mutation and base pair mismatch. [Conclu- sion] The HP intein was rapidly reconstructed by overlap-extension PCR in this study, which not only expands the application of overlap PCR in protein engineering, but also provides a simple, efficient and highly accurate tool for the reconstruction and modification of intein.展开更多
This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated...This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated with Pichia pastoris preferred codon optimization, using the eDNA sequence of tuchyplesin mature pep- tide （54 aa） harboring TAT FFD sequence （11 aa） as reference template, six single-stranded oligonueleotides were designed, the sequences of restriction sites EcoR I and Xba I were introduced to the 5＇ end of primers P1 and P6, respectively. TAT PTD ＋ Tachyplesin fusion gene with a full length of 219 bp was artificially synthesized by overlap extension PCR, which laid the preliminary foundation for subsequent functional and synergism studies.展开更多
基金Supported by National Natural Science Foundation of China(31160032)~~
文摘[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.
基金Supported by the National High Technology Research and Development Program of China(863Program)(2006AA03Z451)Shanghai Basic Research Key Program(10JC1400300)+1 种基金National Natural Science Foundation of China(31070698)the Fundamental Research Funds for the Central Universities(12D10520)~~
文摘[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlapped and served as the template to the other DNA strand in amplification. Then, several rounds of over- lap PCR reaction were conducted to mutate the four cysteines in the HP intein into serines, and introduce a linker sequence containing a tag for product detection and purification. [Result] DNA sequencing analysis proved that the four cysteines in the HP intein were successfully mutated into serines; the PCR precursor fragments were also rejoined into an intact HP gene fragment, without introducing any other unex- pected mutation; the DNA linker of 46 bp was successfully inserted into the de- signed HP gene sequences, without any mutation and base pair mismatch. [Conclu- sion] The HP intein was rapidly reconstructed by overlap-extension PCR in this study, which not only expands the application of overlap PCR in protein engineering, but also provides a simple, efficient and highly accurate tool for the reconstruction and modification of intein.
基金Supported by Natural Science Foundation of Guangdong Province(06025389)Post-doctoral Project of Fujian Province
文摘This study aimed to investigate the synergism of the TAT PTD （ protein transduction domain in HIV-1 transactivator of transcription protein） to antibacte- rial peptide tachyplesin from Tachp/eus tr/dentatus. Treated with Pichia pastoris preferred codon optimization, using the eDNA sequence of tuchyplesin mature pep- tide （54 aa） harboring TAT FFD sequence （11 aa） as reference template, six single-stranded oligonueleotides were designed, the sequences of restriction sites EcoR I and Xba I were introduced to the 5＇ end of primers P1 and P6, respectively. TAT PTD ＋ Tachyplesin fusion gene with a full length of 219 bp was artificially synthesized by overlap extension PCR, which laid the preliminary foundation for subsequent functional and synergism studies.
