In this study, we report an approach to achieve sequence-specific counting of single DNA molecules, which is required for more versatile applications of the previously reported absolute DNA quantification technique based on flow cytometric DNA single molecule counting. While using the same capillary-based flow cytometric setup, fluorescence activation of a target DNA was made with a number of fluorescent oligonucleotide probes of complementary sequences to that of a target DNA. The feasibility of the proposed approach was tested with 7 kb single-strand M13 DNA as the target DNA for sequence specific counting for quantification. Sample preparation, the number of fluorescent oligonucleotide probes, and hybridization conditions mainly matter for the performance of the proposed method. Using a set of 30 sequence-specific fluorescent probes with a selected hybridization buffer, acceptable performance was confirmed through comparison with other conventional methods such as digital polymerase chain reaction (dPCR), UV spectrophotometry, and deoxyribonucleoside monophosphate analysis by mass spectrometry. Proven comparability to the dPCR method confirmed the feasibility of the proposed approach. With further improvement in instrumentation, the proposed method is expected to become established as a reference measurement procedure for sequence-specific quantification of nucleic acids working under a uniquely straightforward measurement principle.

在这项研究中，我们报告了一种实现对单个 DNA 分子进行序列特异性计数的方法，这是先前报道的基于流式细胞术 DNA 单分子计数的绝对 DNA 定量技术的更通用应用所必需的。在使用相同的基于毛细管的流式细胞仪装置时，目标 DNA 的荧光激活是用与目标 DNA 序列互补的许多荧光寡核苷酸探针进行的。以 7 kb 单链 M13 DNA 作为目标 DNA 进行序列特异性计数以进行量化测试了所提出方法的可行性。样品制备、荧光寡核苷酸探针的数量和杂交条件主要影响所提出方法的性能。使用一组 30 个序列特异性荧光探针和选定的杂交缓冲液，通过与其他常规方法（如数字聚合酶链式反应 (dPCR)、紫外分光光度法和质谱法分析脱氧核糖核苷单磷酸盐）进行比较，确认了可接受的性能。经证明与 dPCR 方法的可比性证实了所提出方法的可行性。随着仪器的进一步改进，所提出的方法有望成为在独特的直接测量原理下工作的核酸序列特异性定量的参考测量程序。和脱氧核糖核苷一磷酸通过质谱分析。经证明与 dPCR 方法的可比性证实了所提出方法的可行性。随着仪器的进一步改进，所提出的方法有望成为在独特的直接测量原理下工作的核酸序列特异性定量的参考测量程序。和脱氧核糖核苷一磷酸通过质谱分析。经证明与 dPCR 方法的可比性证实了所提出方法的可行性。随着仪器的进一步改进，所提出的方法有望成为在独特的直接测量原理下工作的核酸序列特异性定量的参考测量程序。