Mutational spectrometry without phenotypic selection: Human mitochondrial DNA
Mutational spectrometry without phenotypic selection: Human mitochondrial DNA
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DOI：
10.1093/nar/25.4.685
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发表时间：
1997-02-15
影响因子：
14.9
通讯作者：
Thilly, WG
中科院分区：
文献类型：
--
作者：
Khrapko, K;Coller, H;Thilly, WG
通过首先根据它们的解链温度将突变体与非突变体DNA序列分离，然后通过高保真DNA扩增增加拷贝数，我们开发了一种方法，该方法允许在10（-6）或以上的分数处观察生物样品中的点突变。使用这种方法，我们已经观察到在培养的细胞和人体组织样品中线粒体基因组的100个碱基对中的热点点突变，到目前为止，已经分离出19个突变体，它们的分数从4 × 10（-4）下降到检测限，我们进行了特定的测试以确定所观察到的信号是否是由污染引起的伪影，我们还测试了DNA复制错配中间体或最初存在于细胞中的内源性DNA加合物在我们的分离步骤中与真正的突变体一起被包括并在PCR期间转化为突变体的可能性，我们发现，虽然大多数突变体在细胞中表现为双链点突变体，但有些似乎至少部分来自错配中间体或细胞DNA加合物，因此，该技术足以观察人类线粒体DNA中点突变的谱，并且是发现这些突变的主要原因的工具。
By first separating mutant from nonmutant DNA sequences on the basis of their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that allows observation of point mutations in biological samples at fractions at or above 10(-6). Using this method, we have observed the hotspot point mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues, To date, 19 mutants have been isolated, their fractions ranging from 4 x 10(-4) down to the limit of detection, We performed specific tests to determine if the observed signals were artefacts arising from contamination, polymerase errors during PCR or DNA adducts created during the procedure, We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, were included with true mutants in our separation Steps and converted to mutants during PCR, We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at least in part from mismatch intermediates or cellular DNA adducts, This technology is therefore sufficient for the observation of the spectrum of point mutations' in human mitochondrial DNA and is a tool for discovering the primary causes of these mutations.