Improved quantitation of HER-2/neu gene copy number in breast tumor-derived DNA samples.

Abstract

A Southern blot-based assay is presented that increases the simplicity and accuracy of the HER-2/neu gene copy number assignment in breast cancer DNA. Genomic DNA was hybridized simultaneously with probes corresponding to portions of HER-2/neu and a single-copy gene, myeloperoxidase. A unique restriction fragment was detected for each gene. The use of DNA probes of similar mass and equal specific activities resulted in a ratio of band intensities on the resultant autoradiograph that reported the ratio of gene copy numbers directly. Patient samples containing amplified levels of the HER-2/neu gene were identified by simple visual inspection of a single autoradiograph. Analysis of breast cancer samples alongside the cell line DNAs, representing a range of HER-2/neu gene copy numbers, permits visual quantitation of the tumors' gene copy numbers. The authors show that the HER-2/neu gene copy number can be determined accurately in marginally degraded DNA, a feature of some clinical samples.