Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner
Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner
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DOI：
10.1016/s1525-1578(10)60006-2
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发表时间：
2005-02-01
影响因子：
4.1
通讯作者：
Krokan, HE
中科院分区：
文献类型：
--
作者：
Akbari, M;Hansen, MD;Krokan, HE
石蜡包埋组织是分子病理学和遗传学研究的重要材料来源。我们使用从福尔马林固定的石蜡包埋胃肿瘤中分离的DNA，对人类尿嘧啶-DNA糖基酶（UNG）基因的一个区域进行突变分析，该区域编码UNG催化结构域，并检测到明显的碱基取代，经过进一步研究，证明这是聚合酶链反应（PCR）产物。我们证明，PCR中低DNA模板输入可以以序列依赖的方式产生假突变，主要是鸟嘌呤到腺嘌呤的转变。其中一个这样的突变与先前报道的人类神经胶质瘤中UNG基因的突变相同。这种现象不是由样品材料的微观异质性引起的，因为在均匀的稀释质粒扩增后可以看到相同的伪影。我们在16个样本中没有观察到UNG基因的真正突变。我们的研究结果表明，在使用少量模板DNA解释基于pcr的体细胞突变分析数据时应谨慎，并且用于丰富样品材料中假定的突变分子亚群的方法本质上可能是对序列依赖性pcr生成的伪影的进一步扩增。
Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.