Enzymatic deamination of cytidines in DNA is an intrinsic component of antibody maturation and retroviral resistance, but can also be a source of HIV drug resistance and cancer-causing mutations. Here, we developed a high-throughput method based on high resolution melt (HRM) analysis called HyperHRM that can screen genomic DNA for rare hypermutated proviral sequences and accurately quantify the number of C-to-T or G-to-A mutations in each sequence. We demonstrate the effectiveness of the approach by profiling in parallel the intensity of the DNA mutator activity of all seven human APOBEC3 proteins on the near full-length sequence of HIV-1 and the Moloney murine leukemia virus. Additionally, HRM was successfully used to identify hypermutated proviral sequences in peripheral blood mononuclear cells from an HIV-1 patient. These results exemplify the effectiveness of HRM-based approaches for hypermutation quantification and for the detection of hypermutated DNA sequences potentially associated with disease or retroviral drug resistance.

中文翻译：

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通过 HyperHRM 分析，沿着 HIV-1 和 MoMLV 的近全长基因组对人类 APOBEC3 蛋白活性进行脱氨强度分析。


DNA 中胞苷的酶促脱氨基是抗体成熟和逆转录病毒耐药性的内在组成部分，但也可能是 HIV 耐药性和致癌突变的根源。在这里，我们开发了一种基于高分辨率熔解 (HRM) 分析的高通量方法，称为 HyperHRM，该方法可以筛选基因组 DNA 中罕见的超突变原病毒序列，并准确量化每个基因中 C 到 T 或 G 到 A 突变的数量。顺序。我们通过平行分析所有七种人类 APOBEC3 蛋白对 HIV-1 和莫洛尼鼠白血病病毒的近全长序列的 DNA 突变活性强度，证明了该方法的有效性。此外，HRM 还成功用于鉴定 HIV-1 患者外周血单核细胞中超突变的原病毒序列。这些结果证明了基于 HRM 的超突变定量方法以及检测可能与疾病或逆转录病毒耐药性相关的超突变 DNA 序列的有效性。