Designing primers for PCR-based diagnostics was achieved by executing sight searches on DNA sequences. Visual searching for specific DNA targets is time consuming, subjective and requires optimisation among numerous c...Designing primers for PCR-based diagnostics was achieved by executing sight searches on DNA sequences. Visual searching for specific DNA targets is time consuming, subjective and requires optimisation among numerous candidate primer sets. Several primer design software have been linked to useful bioinformatic packages to speed the development of PCR assays. Despite the software options available, primer design has remained a challenging aspect of incursion responses, biosecurity emergencies and microbial forensic applications. Two surveys were conducted among 45 plant virologists and 21 other plant pathologists during the 7th Australasian Plant Virology Workshop and the 16th Biennial Australasian Plant Pathology Conference in 2006 and 2007, respectively. Results show that most primer design learning occurs scientist to scientist rather than during academic teaching. This tendency matches with 16% of scientists users of PCR, who do not engage in primer design and 25% designing primers only by visual means, combining a pool of 41% who if trained, would likely enhance their performance in primer design. Only 13 out of 58 scientists ranked themselves as experts. Implementing primer design in study programs and regional training will benefit plant pathology and entomology, and the responsiveness and performance of biosecurity and microbial forensics in the South Pacific.展开更多
masD和bamA是控制石油烃厌氧降解的关键基因,利用实时荧光定量PCR技术检测masD和bamA基因具有简便快速和易操作等优点.但目前所用方法存在扩增效率低,方法灵敏度较差的问题.本文根据引物设计原则,利用Allele ID6软件重新设计了扩增masD...masD和bamA是控制石油烃厌氧降解的关键基因,利用实时荧光定量PCR技术检测masD和bamA基因具有简便快速和易操作等优点.但目前所用方法存在扩增效率低,方法灵敏度较差的问题.本文根据引物设计原则,利用Allele ID6软件重新设计了扩增masD和bamA的实时荧光定量PCR引物,将质粒DNA进行8次10倍梯度稀释后构建实时荧光定量PCR标准曲线.优化后的体系(20μL)为:FastStart Essential DNA Green Master 10.0μL,上下游引物各0.4μL,RNase-Free Water 4.2μL,5.0μL DNA模板.利用新设计的引物扩增masD和bamA基因的最适退火温度分别为61℃和57℃.优化后的检测方法扩增效率提高至97.5%和71.2%,比文献报道的方法提高了7.6%—44.5%,具有更高的重复性和灵敏度.利用设计的引物对陕北5个地区石油污染土壤中的masD和bamA基因进行定量检测结果表明,石油污染土壤中普遍存在着控制石油烃厌氧降解的关键基因,所测定的土壤中bamA降解基因的拷贝数远高于masD降解基因.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
文摘Designing primers for PCR-based diagnostics was achieved by executing sight searches on DNA sequences. Visual searching for specific DNA targets is time consuming, subjective and requires optimisation among numerous candidate primer sets. Several primer design software have been linked to useful bioinformatic packages to speed the development of PCR assays. Despite the software options available, primer design has remained a challenging aspect of incursion responses, biosecurity emergencies and microbial forensic applications. Two surveys were conducted among 45 plant virologists and 21 other plant pathologists during the 7th Australasian Plant Virology Workshop and the 16th Biennial Australasian Plant Pathology Conference in 2006 and 2007, respectively. Results show that most primer design learning occurs scientist to scientist rather than during academic teaching. This tendency matches with 16% of scientists users of PCR, who do not engage in primer design and 25% designing primers only by visual means, combining a pool of 41% who if trained, would likely enhance their performance in primer design. Only 13 out of 58 scientists ranked themselves as experts. Implementing primer design in study programs and regional training will benefit plant pathology and entomology, and the responsiveness and performance of biosecurity and microbial forensics in the South Pacific.
文摘masD和bamA是控制石油烃厌氧降解的关键基因,利用实时荧光定量PCR技术检测masD和bamA基因具有简便快速和易操作等优点.但目前所用方法存在扩增效率低,方法灵敏度较差的问题.本文根据引物设计原则,利用Allele ID6软件重新设计了扩增masD和bamA的实时荧光定量PCR引物,将质粒DNA进行8次10倍梯度稀释后构建实时荧光定量PCR标准曲线.优化后的体系(20μL)为:FastStart Essential DNA Green Master 10.0μL,上下游引物各0.4μL,RNase-Free Water 4.2μL,5.0μL DNA模板.利用新设计的引物扩增masD和bamA基因的最适退火温度分别为61℃和57℃.优化后的检测方法扩增效率提高至97.5%和71.2%,比文献报道的方法提高了7.6%—44.5%,具有更高的重复性和灵敏度.利用设计的引物对陕北5个地区石油污染土壤中的masD和bamA基因进行定量检测结果表明,石油污染土壤中普遍存在着控制石油烃厌氧降解的关键基因,所测定的土壤中bamA降解基因的拷贝数远高于masD降解基因.
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.
