基因改造玉米DNA之萃取與參考質體定量檢測方法之探討

Studies on DNA Extraction and Quantitation Detection of Genetically Modified Maize Using Reference Plasmid

陳任道(Jen-Tao Chen) ； 溫秋燕(Chiou-Yen Wen) ； 趙梅琍(Mei-Li Chao) ； 朱文深(Wen-Shen Chu)

《臺灣農業化學與食品科學》 43卷2期 (2005/04) Pp. 139-148

https://doi.org/10.6578/TJACFS.2005.019

摘要

In quantitative detection of genetically modified maize using reference plasmid, a coefficient k (internal standard ratio) is used to calculate the GMO content of maize. In this study, two factors, the quality of the extracted maize DNA and the reference plasmid, were found to affect significantly the k value. The quality of the extracted maize DNA could be greatly improved by incorporation of α-amylase and proteinase K during extraction. The modified QIAGEN DNeasy Plant Mini kit method developed in this study was easy to operate and high quality maize DNA could be obtained in less than three hours. The commercial reference plasmid is linear and was found to be unstable. The standard curve generated by plotting Ct value against log [copy number] had an R^2 value less than 0.97. A circular reference plasmid pTSSBt was constructed by ligation of seven specific maize transgenic gene fragments and one species gene (SSII) generated by PCR. Standard curve with an R^2 value higher than 0.998 was obtained by using the circular reference plasmid.

關鍵字

基因改造玉米 ； 萃取方法 ； 參考質體 ； 定量PCR ； 內標比係數

並列摘要

In quantitative detection of genetically modified maize using reference plasmid, a coefficient k (internal standard ratio) is used to calculate the GMO content of maize. In this study, two factors, the quality of the extracted maize DNA and the reference plasmid, were found to affect significantly the k value. The quality of the extracted maize DNA could be greatly improved by incorporation of α-amylase and proteinase K during extraction. The modified QIAGEN DNeasy Plant Mini kit method developed in this study was easy to operate and high quality maize DNA could be obtained in less than three hours. The commercial reference plasmid is linear and was found to be unstable. The standard curve generated by plotting Ct value against log [copy number] had an R^2 value less than 0.97. A circular reference plasmid pTSSBt was constructed by ligation of seven specific maize transgenic gene fragments and one species gene (SSII) generated by PCR. Standard curve with an R^2 value higher than 0.998 was obtained by using the circular reference plasmid.

並列關鍵字

GM maize ； DNA extraction method ； Reference plasmid ； Real time PCR ； Internal standard ratio (k)