Rapid detection of MYCN gene amplification in neuroblastomas using the polymerase chain reaction.
We have used the polymerase chain reaction (PCR) to detect amplification of the MYCN oncogene in neuroblastoma cell lines and to distinguish primary tumors with a single copy from those with MYCN amplification using DNA extracted from frozen sections. Two primer pairs were used to co-amplify a 428-bp fragment of the MYCN oncogene along with a 268-bp fragment of the beta-globin gene (a single-copy reference standard). After 30 cycles of PCR, the products were resolved by agarose gel electrophoresis. MYCN gene amplification was identified by visual comparison of the relative intensities of MYCN and beta-globin PCR product bands on the ethidium bromide-stained gel. This semiquantitative approach, while inadequate for precise determination of copy number, provided a simple, rapid, nonisotopic method for differentiating tumors with MYCN amplification from those with a single copy. Seventy-four primary tumors were classified as amplified or nonamplified by semiquantitative PCR. Twenty-two of 23 tumors known to carry MYCN gene amplification by Southern analysis were correctly identified by PCR. The single false-negative result was due to a sampling error: DNA was extracted from a block of tissue containing small foci of tumor surrounded by normal tissue. Fifty-one of 51 tumors with a single copy of MYCN were also correctly identified by PCR. We conclude that semiquantitative PCR is a reliable, non-isotopic alternative to Southern blotting for detection of MYCN gene amplification that can be performed rapidly on DNA extracted from frozen sections.
Crabbe
D C
DC
Department of Pediatrics, Children's Hospital Los Angeles, CA 90054-0700.
Peters
J
J
Seeger
R C
RC
D001483:Base Sequence; D015139:Blotting, Southern; D002648:Child; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005069:Evaluation Studies as Topic; D005784:Gene Amplification; D016259:Genes, myc; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000378:metabolism
D017931:DNA Primers; D004273:DNA, Neoplasm