Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthes...Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.展开更多
【目的】深化柑橘减数分裂粗线期染色体结构组成和配对分析。【方法】首次利用柑橘染色体全长特异寡核苷酸荧光原位杂交(oligo-fluorescence in situ hybridization,oligo-FISH)涂染探针,精准示踪鉴定沙田柚粗线期1号染色体的结构特征,...【目的】深化柑橘减数分裂粗线期染色体结构组成和配对分析。【方法】首次利用柑橘染色体全长特异寡核苷酸荧光原位杂交(oligo-fluorescence in situ hybridization,oligo-FISH)涂染探针,精准示踪鉴定沙田柚粗线期1号染色体的结构特征,比较其与有丝分裂间期细胞核、有丝分裂前中期和中期染色体的结构差异,并揭示粗线期全长染色体的同源配对。【结果】观测到同源配对的全长粗线期与有丝分裂中期较大染色质结构差异。经测定,柚粗线期1号染色体平均全长、长臂长、短臂长和臂比分别为中期的13.93倍、16.54倍、10.44倍和1.54倍,表现出更高FISH信号分辨率。检测到柚粗线期1号染色体着丝粒附近约3.01 Mb的探针信号缺失区。【结论】首次实现柑橘特定全长粗线期染色体的跟踪研究,为深入开展其结构和配对遗传利用的精准研究提供了新方法。展开更多
Thermo-responsive random copolymers,poly(2-(2-methoxyethoxy)ethoxyethyl methacrylate-co-(ethylene glycol)methyl ether methacrylate)(P(EO_(2)-co-EO_(4/5)))and poly(2-(2-methoxyethoxy)ethoxyethyl methacrylate-co-ethylen...Thermo-responsive random copolymers,poly(2-(2-methoxyethoxy)ethoxyethyl methacrylate-co-(ethylene glycol)methyl ether methacrylate)(P(EO_(2)-co-EO_(4/5)))and poly(2-(2-methoxyethoxy)ethoxyethyl methacrylate-co-ethylene glycol methacrylate(P(EO2-co-EG4/5))are synthesized via atom transfer radical polymerization(ATRP).The successful synthesis and the narrow polydispersity index(PDI)of two copolymers are indicated by 1H nuclear magnetic resonance(1H-NMR)and gel permeation chromatography(GPC)analyses.The transition behaviors of polymers in the aqueous solution are demonstrated by changes in turbidity and particle sizes.The transition behavior of P(EO2-co-EG4/5)is found to be milder than that of P(EO2-co-EO4/5).Moreover,the presence of hydrogen bonds without thermo-responsive properties established by hydroxyl groups in the end-side chain of P(EO_(2)-co-EG_(4/5))hinders the dehydration at the transition temperature(TT).Attenuated total reflection Fourier transform infrared spectrometry(ATR-FTIR)analysis along with contact angle measurements reveals that both P(EO_(2)-co-EO_(4/5))and P(EO_(2)-co-EG_(4/5))films undergo phase transitions from hydrophilicity to hydrophobicity above TT.By examining the swelling and collapse behaviors of the polymer films during phase transitions,it can be concluded that the end hydroxyl groups may establish hydrogen bonds with neighboring ether groups within the films,which remain intact throughout the phase transition process due to their strong bonding interactions.This leads to an increase in steric hindrance within swollen films thereby impeding dehydration processes and inducing hysteresis during phase transitions.展开更多
为给梨品种S基因芯片的制备及梨品种自交不亲和性S-RNase基因和S基因型的检测奠定基础,结合已有的对梨自交不亲和S-RNase基因的研究成果及Genbank中梨S-RNase基因信息,利用生物学比对软件Genedoce对梨S-RNase等位基因序列进行比对,得到...为给梨品种S基因芯片的制备及梨品种自交不亲和性S-RNase基因和S基因型的检测奠定基础,结合已有的对梨自交不亲和S-RNase基因的研究成果及Genbank中梨S-RNase基因信息,利用生物学比对软件Genedoce对梨S-RNase等位基因序列进行比对,得到等位基因HV区的特异序列;用oligo6.71、Premier Premier 5.0和Clustal.W等生物学软件设计特异性高、解链温度Tm值接近、长度均一的oligo探针,获得了24条32 mer的oligo探针。展开更多
目的设计单纯疱疹病毒2型(HSV-2)诊断芯片的O ligo探针。方法利用BLA ST软件对HSV-2的DNA序列进行序列比对,得到有意义的特异序列;用生物学软件O ligo6.0设计特异性高、Tm值相近、长度均一的O ligo探针。结果获得13条60-m er O ligo探...目的设计单纯疱疹病毒2型(HSV-2)诊断芯片的O ligo探针。方法利用BLA ST软件对HSV-2的DNA序列进行序列比对,得到有意义的特异序列;用生物学软件O ligo6.0设计特异性高、Tm值相近、长度均一的O ligo探针。结果获得13条60-m er O ligo探针。结论利用BLA ST系统和生物学软件O ligo6.0设计HSV-2诊断芯片的探针,可为后期打印成DNA芯片,用于HSV-2的检测打下基础。展开更多
文摘针对Oligo(d T)亲和层析介质的吸附性能,以poly(A)为模型分子,考察了4种Oligo(d T)亲和层析介质的静态吸附平衡、吸附动力学和动态结合载量(DBC),探讨了载量影响相关机制。结果表明,4种介质的合适吸附条件均为0.6 mol·L-1Na Cl、p H=6~7;Monomix d T20静态吸附容量最大,且poly(A)能扩散至介质微球深层孔内,而Poros Oligo(d T)25、Praesto Jetted (d T)25和Nano Gel d T20等3种介质中poly(A)均主要为表层吸附、静态吸附容量稍低;对于DBC,Nano Gel d T20和Monomix d T20的10%穿透的DBC较高,而Poros Oligo (d T)25和Praesto Jetted (d T)25相对略低。经分析,影响载量的主要因素包含基质种类、微球孔径、配基密度、间隔臂和配基长度等。对于基质种类,聚苯乙烯基质可能孔道结构较为特别。对于微球孔径,应针对不同大小的m RNA分子定制不同孔径的微球,以平衡传质阻力与可及吸附表面积之间的矛盾,从而增大DBC。
文摘Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.
文摘【目的】深化柑橘减数分裂粗线期染色体结构组成和配对分析。【方法】首次利用柑橘染色体全长特异寡核苷酸荧光原位杂交(oligo-fluorescence in situ hybridization,oligo-FISH)涂染探针,精准示踪鉴定沙田柚粗线期1号染色体的结构特征,比较其与有丝分裂间期细胞核、有丝分裂前中期和中期染色体的结构差异,并揭示粗线期全长染色体的同源配对。【结果】观测到同源配对的全长粗线期与有丝分裂中期较大染色质结构差异。经测定,柚粗线期1号染色体平均全长、长臂长、短臂长和臂比分别为中期的13.93倍、16.54倍、10.44倍和1.54倍,表现出更高FISH信号分辨率。检测到柚粗线期1号染色体着丝粒附近约3.01 Mb的探针信号缺失区。【结论】首次实现柑橘特定全长粗线期染色体的跟踪研究,为深入开展其结构和配对遗传利用的精准研究提供了新方法。
基金Fujian External Cooperation project of Natural Science Foundation,China(No.2022I0042)。
文摘Thermo-responsive random copolymers,poly(2-(2-methoxyethoxy)ethoxyethyl methacrylate-co-(ethylene glycol)methyl ether methacrylate)(P(EO_(2)-co-EO_(4/5)))and poly(2-(2-methoxyethoxy)ethoxyethyl methacrylate-co-ethylene glycol methacrylate(P(EO2-co-EG4/5))are synthesized via atom transfer radical polymerization(ATRP).The successful synthesis and the narrow polydispersity index(PDI)of two copolymers are indicated by 1H nuclear magnetic resonance(1H-NMR)and gel permeation chromatography(GPC)analyses.The transition behaviors of polymers in the aqueous solution are demonstrated by changes in turbidity and particle sizes.The transition behavior of P(EO2-co-EG4/5)is found to be milder than that of P(EO2-co-EO4/5).Moreover,the presence of hydrogen bonds without thermo-responsive properties established by hydroxyl groups in the end-side chain of P(EO_(2)-co-EG_(4/5))hinders the dehydration at the transition temperature(TT).Attenuated total reflection Fourier transform infrared spectrometry(ATR-FTIR)analysis along with contact angle measurements reveals that both P(EO_(2)-co-EO_(4/5))and P(EO_(2)-co-EG_(4/5))films undergo phase transitions from hydrophilicity to hydrophobicity above TT.By examining the swelling and collapse behaviors of the polymer films during phase transitions,it can be concluded that the end hydroxyl groups may establish hydrogen bonds with neighboring ether groups within the films,which remain intact throughout the phase transition process due to their strong bonding interactions.This leads to an increase in steric hindrance within swollen films thereby impeding dehydration processes and inducing hysteresis during phase transitions.
文摘为给梨品种S基因芯片的制备及梨品种自交不亲和性S-RNase基因和S基因型的检测奠定基础,结合已有的对梨自交不亲和S-RNase基因的研究成果及Genbank中梨S-RNase基因信息,利用生物学比对软件Genedoce对梨S-RNase等位基因序列进行比对,得到等位基因HV区的特异序列;用oligo6.71、Premier Premier 5.0和Clustal.W等生物学软件设计特异性高、解链温度Tm值接近、长度均一的oligo探针,获得了24条32 mer的oligo探针。
文摘目的设计单纯疱疹病毒2型(HSV-2)诊断芯片的O ligo探针。方法利用BLA ST软件对HSV-2的DNA序列进行序列比对,得到有意义的特异序列;用生物学软件O ligo6.0设计特异性高、Tm值相近、长度均一的O ligo探针。结果获得13条60-m er O ligo探针。结论利用BLA ST系统和生物学软件O ligo6.0设计HSV-2诊断芯片的探针,可为后期打印成DNA芯片,用于HSV-2的检测打下基础。
