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

Abstract

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.