Rapid detection of MYCN gene amplification in neuroblastomas using the polymerase chain reaction.
Rapid detection of MYCN gene amplification in neuroblastomas using the polymerase chain reaction.
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DOI：
10.1097/00019606-199212000-00002
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发表时间：
1992-12-01
期刊：
影响因子：
--
通讯作者：
Seeger, R C
中科院分区：
文献类型：
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作者：
Crabbe, D C;Peters, J;Seeger, R C
我们已经使用聚合酶链反应（PCR）检测扩增的MYCN癌基因在神经母细胞瘤细胞系，并区分原发性肿瘤与单拷贝与MYCN扩增使用从冷冻切片提取的DNA。使用两个引物对共扩增MYCN癌基因的428-bp片段沿着β-珠蛋白基因的268-bp片段（单拷贝参考标准）。在30个PCR循环后，通过琼脂糖凝胶电泳解析产物。通过目视比较溴化乙锭染色凝胶上MYCN和β-珠蛋白PCR产物条带的相对强度来鉴定MYCN基因扩增。这种半定量方法虽然不足以精确测定拷贝数，但提供了一种简单、快速、非同位素的方法，用于区分MYCN扩增的肿瘤与单拷贝的肿瘤。通过半定量PCR将74个原发性肿瘤分类为扩增或非扩增。通过Southern分析已知携带MYCN基因扩增的23个肿瘤中的22个通过PCR正确鉴定。单一的假阴性结果是由于采样错误：DNA是从含有被正常组织包围的小肿瘤病灶的组织块中提取的。51例MYCN单拷贝肿瘤中的51例也通过PCR正确鉴定。我们的结论是，半定量PCR是一种可靠的，非同位素替代Southern印迹检测MYCN基因扩增，可以快速进行从冷冻切片提取的DNA。
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.