Detection of Segregation of Foreign Genes in Transgenic Wheat Lines by Multiplex Polymerase Chain Reaction / 中国生物工程杂志

ABSTRACT

The method of multiplex PCR was set up to identify two or three transgenes in one reaction such as uidA and bar; uidA and IDx5 or uidA, bar and 1Dx5 genes. Three sets of primer pairs which was specific to each of these three genes respectively were designed and synthesized. Recombinant plasmids pAHC25 and p1Dx5 harboring uidA + bar and 1Dx5 gene separately were used as template DNA in the process of optimizing an multiplex PCR reaction. The optimal annealing temperature for uidA and bar MPCR is range from 57. 1℃-62. 3℃ , for uidA and 1Dx5 is range from 60℃ to 60. 6℃ , and for uidA、bar and 1Dx5 range from 57. 0℃-58. 4℃. The amount of template for MPCR is twice as much as that for simplex PCR, while the concentration of primers is the same with simplex one. Less than 50bp MPCR products can be separated clearly by 10% non-denaturalized polyacrylamid gel electrophoresis. Fourteen transgenic wheat lines were tested by multiplex and simplex PCR respectively, which shows the same results and hence presents that MPCR is the reliable, rapid and high-effective approach to detect foreign genes from transgenic plant.