Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic

来源 :Journal of Northeast Agricultural University(English Edition | 被引量 : 0次 | 上传用户:yelangqishi
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TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to 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. The standard curve equation of endogenous reference gene was y =-3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =-3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy. TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV 35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to 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 power fitting-gain Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y = -3.422x + 35.201, R2 = 0.998; the standard curve equation of exogenous gene was y = -3.495x + 35.303, R2 = 0.999. The sample copies were got by putting Ct value into the standard curve equation, and was was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.
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