Establishment of a competitive PCR for N-myc amplification in neuroblastoma

N-myc基因扩增与神经母细胞瘤预后不良密切相关。虽然神经母细胞瘤中N-myc扩增的检测非常重要，但目前仅采用Southern杂交法存在着耗时长、所需DNA量大、操作复杂等缺点。为了克服这些缺点，我们计划将竞争性聚合酶链式反应(CPCR)应用于N-myc扩增的检测。虽然标准的聚合酶链式反应(PCR)基本上不适合于定量检测给定的模板DNA，但它是对给定模板DNA的剂量的绝对测定。1995年，我们利用竞争质粒pZH2建立了测定N-myc扩增的CPCR体系。1996年，我们利用少量样本检验了CPCR在拷贝数测定中的有效性。我们检测了6例神经母细胞瘤针吸活检标本中N-myc基因的拷贝数，验证了其有效性。综上所述，本研究建立的CPCR是一种快速、简便、适用于小样本的方法，可作为Southern杂交检测N-myc拷贝数的一种替代方法。

N-myc amplification correlates well with the poor prognosis of neuroblastoma. Although the detection of N-myc amplification in neuroblastoma is of great importance, Southern hybridization, being employed solely so far, has some disadvantages such as time consuming, the requirement of larger amount of DNA,and somewhat complicated procedure. In order to overcome the disadvantages, we planned to apply competitive PCR (cPCR) to the measurement of N-myc amplification. Although the standard polymerase chain reaction (PCR) is essentially unsuitable for the quantitative detection of a given template DNA,cPCR is an absolute determination of the dose of a given template DNA.In 1995, we established cPCR system for determining N-myc amplification by employing the competitor plasmid pZH2.In 1996, we examined the availability of cPCR in the copy-number determination using the samples with small amounts. We tested the N-myc copy number in 6 neuroblastoma samples collected by needle biopsy and demonstrated the availability. Taken together, we concluded cPCR established in this study is a rapid, simple, and being applicable to small samples and can be an alternate method of Southern hybridization to determine N-myc copy number.