Mutational spectrometry: a general approach for hot-spot point mutations in selectable genes.
Mutational spectrometry: a general approach for hot-spot point mutations in selectable genes.
复制标题
突变光谱测定：选择性基因热点点突变的通用方法。
DOI：
10.1073/pnas.89.10.4623
复制
发表时间：
1992
影响因子：
11.1
通讯作者：
Thilly,WG
中科院分区：
文献类型：
--
作者：
Keohavong,P;Thilly,WG
为了观察高精度的点突变谱，用诱变剂苯并[a]芘二醇环氧化物处理独立的人淋巴母细胞样细胞大培养物，并通过6-硫鸟嘌呤抗性选择HPRT基因突变体。每个实验平均产生1.6 × 10(4)个6-硫鸟嘌呤抗性突变体，利用高保真聚合酶链反应和变性梯度凝胶电泳检测HPRT基因外显子3中最常见突变的种类、位置和数量。发现16个外显子3特异性突变主要是G----T翻转，对应于每个实验平均3500个诱导突变。在这些突变中，6个发生在6个鸟嘌呤序列中，5个发生在5‘-GAAGAG-3’序列中。独立实验之间的差异与数值预期一致，即所有16个突变都满足突变热点的合理统计标准。通过对不同系统的数据进行克隆分析，结果表明本文所报道的方案可以成为研究存在表型选择系统的DNA序列的热点点突变谱的有用工具。
To observe point mutational spectra with a high degree of precision, independent large cultures of human lymphoblastoid cells were treated with a mutagen, benzo[a]pyrene diol epoxide, and mutants at the HPRT gene were selected en masse by 6-thioguanine resistance. An average of 1.6 x 10(4) 6-thioguanine-resistant mutants were created per experiment and the kinds, positions, and numbers of the most frequent mutations were examined in exon 3 of the HPRT gene by using a high-fidelity polymerase chain reaction and denaturing gradient gel electrophoresis. Sixteen exon 3-specific mutations were found to be predominantly G----T transversions and corresponded to an average of 3500 induced mutants per experiment. Of these mutations, 6 occurred within a run of 6 guanines and 5 occurred in the sequence 5'-GAAGAG-3'. The variation among independent experiments is consistent with the numerical expectation that all 16 mutations fulfill reasonable statistical criteria for mutational hot spots. The agreement with data from various systems using clone-by-clone analysis shows that the protocol reported herein can be a useful tool to study hot-spot point mutational spectra for DNA sequences for which phenotypic selection systems exist.