Determination of N-myc gene amplification in neuroblastoma by differential polymerase chain reaction.
Determination of N-myc gene amplification, a powerful prognostic indicator in the childhood tumour, neuroblastoma, has routinely been performed by Southern analysis. We have developed a differential polymerase chain reaction (PCR) assay, in which the N-myc target gene is co-amplified with a control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Following electrophoresis, a ratio between the two PCR products within a given DNA sample is then determined by densitometry. This assay was applied to DNA isolated from 32 primary neuroblastoma tumours for which the N-myc status had previously been determined by Southern analysis. Following PCR, samples containing a single copy of the N-myc oncogene were clearly distinguishable from samples with N-myc gene amplification, based on an N-myc/GAPDH ratio of below or above 1.0, respectively. Linear regression indicated a highly significant relationship (R = 0.94; P < 0.0001) between N-myc copy number (Southern) and N-myc/GAPDH ratio (PCR). Serial dilution of N-myc amplified DNA with non-amplified control DNA indicated that the PCR assay was sufficiently sensitive to detect two-fold amplification. Moreover, such serial dilution allowed determination of N-myc copy number. The assay, which requires only small amounts of tissue and does not utilize 32P-radioactivity, therefore provides a rapid and sensitive alternative to Southern analysis.
Gilbert
J
J
Children's Leukaemia and Cancer Research Centre, Prince of Wales Children's Hospital, Randwick, Sydney, N.S.W., Australia.
Norris
M D
MD
Haber
M
M
Kavallaris
M
M
Marshall
G M
GM
Stewart
B W
BW
D001483:Base Sequence; D015139:Blotting, Southern; D002648:Child; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D005784:Gene Amplification; D016259:Genes, myc / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases