Quantitation of erbB-2 gene copy number in breast cancer by an improved polymerase chain reaction (PCR) technique, competitively differential PCR

A new method of measuring gene copy number in small samples of DNA was used to measure amplification of the erbB-2 gene and a reference gene in breast cancers. The method, termed 'competitively differential polymerase chain reaction' (CD-PCR), combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR (D-PCR) and competitive PCR (C-PCR). The CD-PCR methodology was evaluated for sensitivity and specificity by comparing amplification measured by CD-PCR with that obtained by fluorescence in situ hybridization (FISH), C-PCR, and Southern blotting analysis. CD-PCR analysis proved to be an accurate predictor of amplification. CD-PCR also overcomes the problems involved in variation of PCR efficiencies and DNA concentrations in tumor samples, and the problems caused by the plateau effect in PCR.