Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan.
Int J Mol Sci. 2020 Jul 20;21(14):5119. doi: 10.3390/ijms21145119.
Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method in which PCR amplification of a target sequence is inhibited in a sequence-specific manner by the hybridization of an ORN with the target sequence. Previously, we reported that ORNi-PCR could detect nucleotide mutations in DNA purified from cultured cancer cell lines or genome-edited cells. In this study, we investigated whether ORNi-PCR can discriminate nucleotide differences and CpG methylation status in damaged DNA, such as tissue specimen DNA and bisulfite-treated DNA. First, we showed that ORNi-PCR could discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded rat liver specimens or formalin-fixed paraffin-embedded human specimens. Rat whole blood specimens were compatible with ORNi-PCR for the same purpose. Next, we showed that ORNi-PCR could discriminate CpG methylation status in bisulfite-treated DNA. These results demonstrate that ORNi-PCR can discriminate nucleotide differences and CpG methylation status in multiple types of DNA samples. Thus, ORNi-PCR is potentially useful in a wide range of fields, including molecular biology and medical diagnosis.
寡核苷酸 (ORN) 干扰-PCR (ORNi-PCR) 是一种方法，其中 ORN 与靶序列杂交以序列特异性方式抑制靶序列的 PCR 扩增。以前，我们报道过 ORNi-PCR 可以检测从培养的癌细胞系或基因组编辑细胞中纯化的 DNA 中的核苷酸突变。在这项研究中，我们研究了 ORNi-PCR 是否可以区分受损 DNA（如组织标本 DNA 和亚硫酸氢盐处理的 DNA）中的核苷酸差异和 CpG 甲基化状态。首先，我们表明 ORNi-PCR 可以区分从丙酮固定的石蜡包埋大鼠肝标本或福尔马林固定的石蜡包埋人标本中提取的 DNA 中的核苷酸差异。大鼠全血标本也适用于 ORNi-PCR 进行相同的目的。接下来，我们表明 ORNi-PCR 可以区分亚硫酸氢盐处理的 DNA 中的 CpG 甲基化状态。这些结果表明 ORNi-PCR 可以区分多种类型 DNA 样本中的核苷酸差异和 CpG 甲基化状态。因此，ORNi-PCR 在包括分子生物学和医学诊断在内的广泛领域具有潜在的用途。
