多重PCR法快速鉴定转基因小麦植株及后代

Rapid confirmation of the transgenic wheat lines by multiplex polymerase chain reaction (MPCR)

根据转基因植物中常用的报告基因uidA、选择基因bar的序列 ,设计合成两对不同的引物 ,建立了在转基因小麦材料分子确证中 ,应用一次PCR反应同时扩增检测两种或多种外源基因的多重PCR方法 .通过对质粒 pAHC2 5以及 30个转基因小麦样品进行PCR扩增分析 ,比较多重PCR的检测结果与单基因的PCR扩增结果 。

A method of multiplex PCR was set up to identify two transgenes uidA and bar in one reaction. According to the specific sequences of uidA and bar , two sets of the primers were designed and synthesized. The plasmid pAHC25 including the genes of uidA and bar was used as template DNA in the process of optimizing a multiplex PCR reaction. It was testified that the two sets of primers were specific and effective. The optimal condition of MPCR for uidA and bar were as follows: the concentration of each primer was 0.4?μmol, the annealing temperature was 58?℃,and the optimal concentration of template DNA was 90?pg/μL. The concentration of dNTPs and the change of cycles did not show the obvious effect on MPCR. Thirty transgenic wheat samples were tested by multiplex and simplex PCR, respectively, and the results were consistent for two target transgenes.