Rapid quantitative analysis of differential PCR products by high-performance liquid chromatography

We describe the use of high-performance liquid chromatography (HPLC) for the rapid quantitative analysis of short DNA fragments generated in differential PCR, where a target gene and a control gene are in vitro coamplified in one single reaction. Using an anion-exchange nonporous column, both separation and quantitation of the differential PCR products are achieved in about 5 min per sample. The performance of this technique proved to be superior to that of conventional gel electrophoresis and subsequent analysis by a laser densitometer, a solid-state scanner and a charge coupled device video camera imaging system. The usefulness for clinical testing is described in the example of the quantitative analysis of the c-erbB-2 oncogene copy number of human breast carcinomas by differential PCR. The combined use of differential PCR and automated HPLC analysis of the PCR products may well substitute for classical Southern blot hybridization in routine clinical analysis of oncogene amplification.