以链霉菌702-20为出发菌株,经HNO2诱变处理,获得高产突变株。实验结果表明:HNO2处理20 m in对菌株的致死率可达83.10%,突变率高达14.13%,经过摇瓶筛选获得高产突变株20-29-148,产链霉素能力达到1.404 mg/mL,比出发菌株提高了37.65%。经...以链霉菌702-20为出发菌株,经HNO2诱变处理,获得高产突变株。实验结果表明:HNO2处理20 m in对菌株的致死率可达83.10%,突变率高达14.13%,经过摇瓶筛选获得高产突变株20-29-148,产链霉素能力达到1.404 mg/mL,比出发菌株提高了37.65%。经传代培养考察,该突变菌株具有良好的遗传稳定性。展开更多
Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1...Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1,2 and 7 bp)and 2 deletions(1 and 2 bp)in the hepatocyte nuclear factor(HNF)-4α,glucokinase and HNF-1α genes were tested.During nested PCR,amplified fragments were labeled with three fluorescent dyes.PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in nondenaturing gel conditions.Results Twenty-five of 27 variants(93%)could be detected by combining 5% glycerol and 10% sucrosegel matrix conditions.Twenty-two of 27(82%)and 18 of 27(67%)variants were identified using 5%glycerol and 10% sucrose conditions,respectively.Conclusion This fluorescence-based PCR single strand conformation polymorphism technique represents a simple,non-hazardous,time-saving and sensitive method for high throughput mutation detection.展开更多
The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics.The immunoprecipitation purification or chemical pulldown technique is ge...The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics.The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs.Here we developed an a^(6)A-seq method,which takes advantage of N^(6)-allyladenosine(a^(6)A)metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner.a^(6)A plays a role as a chemical sequencing tag in that the iodination of a^(6)A in mRNAs results in 1,N^(6)-cyclized adenosine(cyc-A),which induces base misincorporation during RNA reverse transcription,thus making a^(6)A-labelled mRNAs detectable by sequencing.A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine.a^(6)A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition.Compared with regular RNA-seq,a^(6)A-seq could more sensitively detect the change of mRNA production over a time scale.The experiment of a^(6)Acontaining mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a^(6)A-seq data.Together,our results show a^(6)A-seq is an effective tool to study RNA dynamics.展开更多
The suggestion that mutation of mitochondria of cell energy producer is linked to oncogenesis could be traced back at least to 40 years ago. Although there has been plenty of evidence in favour of it, no convincing me...The suggestion that mutation of mitochondria of cell energy producer is linked to oncogenesis could be traced back at least to 40 years ago. Although there has been plenty of evidence in favour of it, no convincing mechanism has even been proposed. Many invesstigators have been interested in a comparatively neglected area of tumor mitochondria, since the existence of mitochondrial DNA (mtDNA) in forms of the ab-展开更多
文摘以链霉菌702-20为出发菌株,经HNO2诱变处理,获得高产突变株。实验结果表明:HNO2处理20 m in对菌株的致死率可达83.10%,突变率高达14.13%,经过摇瓶筛选获得高产突变株20-29-148,产链霉素能力达到1.404 mg/mL,比出发菌株提高了37.65%。经传代培养考察,该突变菌株具有良好的遗传稳定性。
文摘Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1,2 and 7 bp)and 2 deletions(1 and 2 bp)in the hepatocyte nuclear factor(HNF)-4α,glucokinase and HNF-1α genes were tested.During nested PCR,amplified fragments were labeled with three fluorescent dyes.PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in nondenaturing gel conditions.Results Twenty-five of 27 variants(93%)could be detected by combining 5% glycerol and 10% sucrosegel matrix conditions.Twenty-two of 27(82%)and 18 of 27(67%)variants were identified using 5%glycerol and 10% sucrose conditions,respectively.Conclusion This fluorescence-based PCR single strand conformation polymorphism technique represents a simple,non-hazardous,time-saving and sensitive method for high throughput mutation detection.
基金the National Key R&D Program of China(2022YFA1103702 and 2017YFA0506800)the National Natural Science Foundation of China(22022702 and 21977087)the Fundamental Research Funds for the Central Universities,and MOE Key Laboratory of Macromolecular Synthesis and Functionalization,Zhejiang University(2022MSF04).
文摘The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics.The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs.Here we developed an a^(6)A-seq method,which takes advantage of N^(6)-allyladenosine(a^(6)A)metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner.a^(6)A plays a role as a chemical sequencing tag in that the iodination of a^(6)A in mRNAs results in 1,N^(6)-cyclized adenosine(cyc-A),which induces base misincorporation during RNA reverse transcription,thus making a^(6)A-labelled mRNAs detectable by sequencing.A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine.a^(6)A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition.Compared with regular RNA-seq,a^(6)A-seq could more sensitively detect the change of mRNA production over a time scale.The experiment of a^(6)Acontaining mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a^(6)A-seq data.Together,our results show a^(6)A-seq is an effective tool to study RNA dynamics.
文摘The suggestion that mutation of mitochondria of cell energy producer is linked to oncogenesis could be traced back at least to 40 years ago. Although there has been plenty of evidence in favour of it, no convincing mechanism has even been proposed. Many invesstigators have been interested in a comparatively neglected area of tumor mitochondria, since the existence of mitochondrial DNA (mtDNA) in forms of the ab-
