[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DN...[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.[Result] The standard curve equation of endogenous reference gene is y=-3.422x＋35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x＋34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.[Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.展开更多
Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of n...Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of nano Au and TiO2 microsphere was investigated with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored with EIS us-ing [Fe(CN)6]3-/4- as indicator. The sequence-specific DNA of the 35S promoter from cauliflower mosaic virus (CaMV35S) gene was detected with this DNA electrochemical sensor. The dynamic detection range was from 1.0×10-12 to 1.0×10-8 mol/L DNA and a detection limit of 2.3×10-13 mol/L could be ob-tained. The polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase (NOS) gene from the real sample of a kind of transgenic soybean was also satisfactorily detected.展开更多
A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes(PANInt)and poly-L-lysine(pLys).The composite of pLys and PANInt was coated onto the ...A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes(PANInt)and poly-L-lysine(pLys).The composite of pLys and PANInt was coated onto the carbon paste electrode(CPE)to form a uniform and very stable nanocomposite membrane.The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANInt even at physiological pH,but also possesses fine biocompatibility for bio-analytes.DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity.The synergistic effect of PANInt and pLys could significantly enhance the sensitivity of DNA hybridization recognition.The phosphinothricin acetyltransferase(PAT)gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method.This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using[Fe(CN)6]3-/4as an indicator.展开更多
基金Funded by Program of Technology Bureau of Harbin(2010RFQXN101)Sub-project of Transgenic Significant Specific Project(2008ZX08012-001)~~
文摘[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.[Result] The standard curve equation of endogenous reference gene is y=-3.422x＋35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x＋34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.[Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.
基金the National Natural Science Foundation of China (Grant Nos. 20635020 and 20375020)Doctoral Foundation of the Ministry of Education of China (Grant No. 20060426001)Natural Science Foundation of Qingdao City (Grant No. 04-2-JZP-8)
文摘Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of nano Au and TiO2 microsphere was investigated with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored with EIS us-ing [Fe(CN)6]3-/4- as indicator. The sequence-specific DNA of the 35S promoter from cauliflower mosaic virus (CaMV35S) gene was detected with this DNA electrochemical sensor. The dynamic detection range was from 1.0×10-12 to 1.0×10-8 mol/L DNA and a detection limit of 2.3×10-13 mol/L could be ob-tained. The polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase (NOS) gene from the real sample of a kind of transgenic soybean was also satisfactorily detected.
基金the support from the National Natural Science Foundation of China(20635020,20805025&20975057)Doctoral Foundation of the Ministry of Education of China(20060426001)Natural Science Foundation of Qingdao City(09-1-3-25-jch)
文摘A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes(PANInt)and poly-L-lysine(pLys).The composite of pLys and PANInt was coated onto the carbon paste electrode(CPE)to form a uniform and very stable nanocomposite membrane.The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANInt even at physiological pH,but also possesses fine biocompatibility for bio-analytes.DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity.The synergistic effect of PANInt and pLys could significantly enhance the sensitivity of DNA hybridization recognition.The phosphinothricin acetyltransferase(PAT)gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method.This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using[Fe(CN)6]3-/4as an indicator.
