A method for measuring gene copy number in biological samples without using control samples of known copies

Various human diseases are caused by gene copy number variants, underlining the importance of determining disease gene copy number for clinical diagnostics. In this study, we describe a method for determining gene copy number in a biological organism. In this method, both a target gene and an internal control gene were amplified from the organism by regular PCR. The PCR products were purified and quantified, and the target and internal control genes were mixed at different molar ratios. Real-time PCR was then used to measure the quantification cycle (Cq) values of both the target and internal control genes in the mixtures. A standard curve was constructed to correlate the differences between the Cq values and the logarithmic ratios of the target gene to the internal control gene. Real-time PCR was then used to measure the Cq values of both the target and internal control genes in experimental samples; the copy number of the target gene in the sample can be calculated readily from the calculated standard curve. This method was validated by a set of internal control genes and a foreign gene in transgenic alfalfa, demonstrating the utility of this method in the determination of gene copy number for various applications.