Assessment of MYCN amplification in neuroblastoma biopsies by differential polymerase chain reaction.
Amplification of the oncogene MYCN is a genetic change frequently observed in neuroblastoma and is an indicator of poor prognosis. MYCN copy number is currently determined by Southern blot hybridization. This technique takes 2 to 3 weeks, is labor-intensive, is sensitive to DNA degradation, and requires large quantities of DNA. We have evaluated a new, semiquantitative method of estimating gene copy number that uses differential polymerase chain reaction (PCR). The procedure can be performed in 1 day, is highly reproducible, and requires only nanogram quantities of DNA. It employs a semiquantitative, nonisotopic PCR technique based on differential competition for PCR substrates. MYCN gene primers are amplified together with primers from a single-copy internal control gene. Following electrophoretic separation, the ratio of the two PCR products is determined visually and by densitometric analysis of ethidium bromide-stained agarose gels. This differential ratio is then compared to a series of ratios generated from standards of known MYCN gene copy number. We compared the results obtained by this differential PCR method with those obtained by conventional Southern blotting in 16 cases of primary neuroblastoma. All amplified tumors were detected by differential PCR, and no false positives were observed. We confirmed that differential PCR is a rapid and reliable alternative to Southern blotting for MYCN copy number assessment and is highly suited to the analysis of DNA derived from needle biopsies.
Boerner
S
S
Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada.
Squire
J
J
Thorner
P
P
McKenna
G
G
Zielenska
M
M
D001483:Base Sequence; D015139:Blotting, Southern; D004273:DNA, Neoplasm / Q000235:genetics*; D005784:Gene Amplification / Q000235:genetics*; D016259:Genes, myc / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm