Quantitation of c-myc gene amplification by a competitive PCR assay system

Gene amplification is a common event in the progression of human cancers. The detection and quantitation of certain amplified oncogenes has been shown to have prognostic importance in certain human malignancies. A method is described that utilizes the principles of competitive PCR for quantitation of the c-myc gene copy number in relation to the copy number of a reference gene (tissue plasminogen activator [t-PA] gene) located on the same chromosome (8) as the c-myc gene. This ratio gives the true level of amplification of the c-myc gene, accounting for variables such as cell number, cell cycle phase, and chromosome 8 ploidy. The determination of gene amplification depends on the precise measurement of the ratio of target and reference genes. An important feature of this assay is that the competitive reference standards used for target gene c-myc and reference gene t-PA have been linked to form a hybrid. This simple modification guarantees that both reference gene and target gene assay tubes get identical amounts of the competitive template for each gene, thereby eliminating a significant source of error. This method has the same desirable attributes of standard PCR in that very small sample sizes are required and that results can easily be obtained in < 24 hr. In addition, this technique does not require the use of radioactivity or expensive DNA detection kits, and thus, may give it wider applicability for the study of human cancers.