TARGET LENGTH AND PRIMER CONCENTRATION AFFECT THE GAIN OF C-ERBB2 ANDP53 AMPLICONS

Although quantitative polymerase chain reaction (PCR) using internal s tandards may perform reproducibly, the calibration of the normal level is problematic, Three test sequences (171, 213, and 260 base pairs [b p]) from the c-erbB2 oncogene were separately coamplified with a 133 b p 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 s ynthesis of the longer sequence, Increase in primer concentration of t he longer oncogene sequences adjusted this imbalance, and the expected ratio of 1.0 could be reached, The primer or target sequence also inf luenced 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 s table results, probably because of clear band separation in polyacryla mide gel electrophoresis. A ratio estimate of 1.0 was produced by onco gene and control gene printer concentrations of 3.5 pmol/ mu 1 and 0.6 pmol/mu 1, respectively. Calibrated quantitative PCR methodology is a pplicable to many areas and offers an excellent tool for screening all ele deletions, supernumerary alleles, or chromosomes associated with f amilial diseases or disease syndromes.