UV- and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis

Abstract

UV- and visible-excited fluorescence detection strategies were compared for nucleic acids separated by capillary electrophoresis (CE). A dual-polymer sieving matrix consisting of hydroxypropylmethylcellulose and poly(vinylpyrrolidone) was used to separate DNA fragments from a 100-base pair ladder and RNA from individual cells. Two nucleic acid dyes, SYBR Gold and SYBR Green I, were evaluated for their performance at both UV (275 nm) and visible (488 nm) excitation wavelengths. While SYBR Gold-bound RNA from single cells yielded a substantially reduced UV-excited signal compared to that with visible excitation (as expected), the sensitivity of SYBR Gold-bound double-stranded DNA was comparable for UV and Vis excitation wavelengths. This study reveals the first demonstration of using SYBR Gold dyes for DNA detection following separation with CE and also the first example of SYBR-based detection of RNA sampled and separated from individual cells.