Target length and primer concentration affect the gain of c-erbB2 and p53 amplicons

Abstract

Although quantitative polymerase chain reaction (PCR) using internal standards may perform reproducibly, the calibration of the normal level is problematic. Three test sequences (171, 213, and 260 base pairs [bp]) from the c-erbB2 oncogene were separately coamplified with a 133 bp control sequence from the single copy gene p53 in differential PCR. Sequence length differences between the oncogene and control sequences influenced the ratio estimates, obviously because of less efficient synthesis of the longer sequence. Increase in primer concentration of the longer oncogene sequences adjusted this imbalance, and the expected ratio of 1.0 could be reached. The primer or target sequence also influenced the ratio estimate, since the 171 bp oncogene sequence was as efficiently synthesized as the 133 bp control sequence but with lower primer concentration. The 260 bp oncogene sequence produced the most stable results, probably because of clear band separation in polyacrylamide gel electrophoresis. A ratio estimate of 1.0 was produced by oncogene and control gene primer concentrations of 3.5 pmol/microl and 0.6 pmol/microl, respectively. Calibrated quantitative PCR methodology is applicable to many areas and offers an excellent tool for screening allele deletions, supernumerary alleles, or chromosomes associated with familial diseases or disease syndromes.