Direct-Double-Differential PCR for Gene Dosage Quantification of c-myc

c- myc gene amplification that leads to overexpression has been shown to play a major role in cancer development, especially in breast cancer (1). Two methodological approaches based on PCR, differential PCR (2) and competitive PCR (3), have been developed for gene dosage quantification and have been applied to c- myc . When these methods are used, both degraded DNA and the necessarily low competitor concentrations, which are prone to dilution errors, lead to over- or underestimation of gene dosages. Methods using separate comparisons of two single-copy reference genes for any DNA sample (double-differential PCR) (4)(5), dilution series of the competitors (competitive-differential PCR) (6), or sample DNA (in differential PCR) (7) have been introduced to quantitative PCR to circumvent those problems. In consequence, the methods are labor-intensive, time-consuming, and expensive.We improved quantitative PCR and developed a reliable and sensitive direct-double-differential PCR method for c- myc gene dosage quantification in which DNA fragments of two different single-copy reference genes, manganese superoxide dismutase (SOD2) and β-globin (HBB) , and the target DNA fragment (c- myc ) are coamplified simultaneously in one reaction tube. The c- myc PCR product …