Keohavong Phouthone, Xi Liqiang, Grant Stephen G
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA.
Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
Methods Mol Biol. 2020;2102:349-359. doi: 10.1007/978-1-0716-0223-2_20.
The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.
次黄嘌呤磷酸核糖转移酶（HPRT）检测法利用有毒核苷酸类似物的掺入来筛选缺乏嘌呤清除酶——次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶的细胞。该检测法的一个主要优势在于能够分离突变细胞并确定其功能缺陷的分子基础。在这个基因座上已经进行了多种类型的分析：当前的实验方案包括生成互补DNA（cDNA）并对每个外显子进行多重聚合酶链反应（PCR），包括内含子/外显子连接区，随后对产物进行直接测序。这种分析可检测基因内的点突变、小的缺失和插入、影响RNA剪接的突变以及基因内异常V(D)J重组的产物。建立突变谱并进行比较有望从这个易于分析的报告基因处特定基因的DNA损伤独特模式中识别出对诱变基因毒素的暴露情况。
