Multiplex dsDNA Fragment Sizing Using Dimeric Intercalation Dyes and Capillary Array Electrophoresis: Ionic Effects on the Stability and Electrophoretic Mobility of DNA−Dye Complexes

Abstract

Methods have been developed for performing accurate, high-resolution, multiplex capillary electrophoresis separations of dsDNA using dimeric intercalation dyes as noncovalent labeling reagents. The quality of these separations is highly dependent on the cation present during electrophoresis. Using buffers that contain only one cation, we show that the tetrapentylammonium (NPe(4)(+)) ion results in high-resolution, high-sensitivity separations but that smaller ions such as sodium or the commonly used buffer ion tris produce low-resolution, low-intensity separations of DNA-dye complexes. Using an 80 mM taps-NPe(4), 1 mM H(2)EDTA, pH 8.4, 0.8% HEC separation buffer, high-quality multiplex separations were performed using TOTO and buTOTIN, YOYO and TOED2, and TO and buTOTIN labeled restriction digests. In the taps-NPe(4) buffer, there is no significant mobility shift when complexes are formed with DNA-dye ratios from 100 to 5 bp per dye and very little dye transfer was observed. This property permits accurate multiplex sizing of samples having a wide concentration range simply by mixing the DNA with a dye solution before electrophoresis. This capability is demonstrated by diluting unpurified PCR products 10-, 100-, and 1,000-fold before mixing with a 1 nM TOTO solution and separating these samples with a ΦX174 HAEIII sizing ladder complexed with buTOTIN. Sizing precisions of better than 1% were obtained at all concentrations of target DNA. The mechanism for the increased DNA-dye complex stability and electrophoretic resolution in the taps-NPe(4) buffer is discussed.