Fluorescence coupled in-capillary DNA hybridization assay and its application to identification of DNA point mutation

• We examined the ssDNA hybridization inside a capillary.
• We prove CE-FL to be a powerful method to resolve DNA hybridization.
• We achieve chromatographic separation of DNA.
• We discovered a novel strategy for the online detection of base mutation(s).
• The in-capillary assay of integrating multi-steps has been developed.

We have developed a fluorescence coupled in-capillary DNA hybridization assay and applied it to the detection of DNA base pair mutation(s). A 15-mer thrombin-binding single-stranded DNA oligonucleotide labeled with 6-carboxyfluorescein (DNA1-FAM) was mixed and hybridized with its complementary DNA (cDNA1) via the in-capillary approach. A fluorescence coupled capillary electrophoresis (CE-FL) was used to measure the status and reaction rate of DNA double strands hybridization under different reaction conditions, and the results were recorded on the electrophoretograms. Recorded electrophoretograms have shown that the in-capillary assay has different DNA hybridization patterns compared to the post-capillary assay. The DNA hybridization efficiency of the in-capillary assay was affected by the molar ratio, the time interval of injection and the volume of DNA oligos. Interestingly, this assay could be used to detect one or two base pair mutation(s) in the target DNA. The in-capillary assay, characterized by an easy operation, reproducible results, and a low instrumental requirement, can be coupled to an online base pair mutation-based detection system, which would facilitate the related small-scale research analysis at non-major research centers.

This report highlights a fluorescence coupled in-capillary DNA hybridization assay and applied it to the detection of DNA base pair mutation(s). This technique integrated multiple functions “injection, mixing, reaction, separation and detection” in a single capillary. The in-capillary assay can be coupled to an online base pair mutation detection system, which would facilitate the related small-scale research at non-major research centers.Figure optionsDownload as PowerPoint slide