Characterization of the selective staining of DNA on gels using two fluorochromes

Ethidium bromide (EB) and 4'-6-diamidino-2-phenylindole (DAPI) are both well-known fluorochromes for detecting DNA fragments. EB binds to DNA by intercalation and DAPI binds in the DNA minor groove. We previously developed a staining method using both EB and DAPI that is selective for AT-rich DNA fragments. Using this double-staining method, AT-rich DNA fragments are visualized as bluish-white fluorescent bands. To further characterize this method, a series of synthetic DNA fragments were designed with systematic variations in the length, AT content, and DNA sequence pattern. The staining properties of these fragments were determined in the presence of DAPI and EB, and the following results were obtained. (i) In a series of fragments with three AT base pairs followed by one GC base pair, the stained DNA fragments exhibited different fluorescent colors and varied from bluish (more DAPI staining) to pinkish (less DAPI staining) in the order 5'-AAA-3', 5'-AAT-3', 5'-ATA-3', 5'-TTA-3'. (ii) In fragments with constant AT content, the blue fluorescent color increased with increasing number of A (or T) nucleotides, due to increased DAPI binding. The blue color was saturated when the number of A (or T) nucleotides was 12 or greater. (iii) The fluorescent color of the stained DNA fragments changed in the order of red-orange, pink, pinkish-white, white, bluish-white, blue as the AT content increased from 0 to 100%. Thus, the fluorescent color of DNA fragments stained with DAPI and EB depends on base composition and nucleotide sequence, suggesting that individual stained DNA fragments may have characteristic and specific fluorescent colors. The fluorescent color emitted by specific stained DNA fragments in the presence of EB and DAPI can be analyzed with a high degree of sensitivity and resolution using the XYZ colorimetric system.