High-throughput transgene copy number estimation by competitive PCR

Abstract

Transgene copy number affects the level and stability of gene expression. Therefore, it is important to determine the copy number of each transgenic line. Polymerase chain reaction (PCR) is widely employed to quantify amounts of target sequences. Although PCR is not inherently quantitative, various means of overcoming this limitation have been devised. Recent real-time PCR methods are rapid; however, they typically lack a suitable internal standard, limit the size of the target sequence, and require expensive specialized equipment. Competitive PCR techniques avoid these problems, but traditional competitive methods are time consuming. Here we apply mathematical modeling to create a rapid, simple, and inexpensive copy number determination method that retains the robustness of competitive PCR.