High resolution separation of DNA restriction fragments using capillary electrophoresis with near-IR, diode-based, laser-induced fluorescence detection

The near-IR dye thiazole green (TAG) was used as a monomeric nuclear staining dye for the low-level detection of DNA restriction fragments separated via high-performance capillary electrophoresis with near-IR laser-induced fluorescence detection. TAG possessed an absorption maximum at 735 nm and an emission maximum at approximately 765 nm and, in the presence of dsDNAs, showed a fluorescence enhancement ratio of approximately 102, with a binding constant to dsDNAs determined to be 6.1 x 10(6) M(-1). The high-resolution separation of the HaeIII restriction digest of Phi X174 was carried out using capillary electrophoresis on the native, ethidium bromide-stained, and TAG-stained DNA fragments, The TAG-stained DNA fragments resulted in higher plate numbers compared to the native and EtBr-stained restriction fragments as well as enhanced resolution; however, the 271/281 fragments could not be resolved using these CE conditions, To investigate the detection sensitivity of the TAG-stained DNA in capillary electrophoresis, an all-solid-state diode-based, laser-induced fluorescence detector was constructed, which consisted of a GaAlAs diode laser, with a principal lasing line at 750 nm and an avalanche photodiode, Using a running buffer composed of an entangled polymer (HPMC) and 1 mu M TAG with no prestaining of the dsDNA prior to the electrophoresis, the limit of detection was found to be 20 fg (SNR = 3) of DNA per electrophoretic band. In addition, using the LIF system, the 271/281 bp fragments were nearly baseline resolved, with plate numbers exceeding 1 x 10(6) plates/m.