Quantification of single-strand DNA by sequence-specific counting in capillary flow cytometry

Abstract

Abstract 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.