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 s taining dye for the low-level detection of DNA restriction fragments s eparated via high-performance capillary electrophoresis with near-IR l aser-induced fluorescence detection. TAG possessed an absorption maxim um at 735 nm and an emission maximum at approximately 765 nm and, in t he presence of dsDNAs, showed a fluorescence enhancement ratio of appr oximately 102, with a binding constant to dsDNAs determined to be 6.1 x 10(6) M(-1). The high-resolution separation of the HaeIII restrictio n digest of Phi X174 was carried out using capillary electrophoresis o n the native, ethidium bromide-stained, and TAG-stained DNA fragments, The TAG-stained DNA fragments resulted in higher plate numbers compar ed to the native and EtBr-stained restriction fragments as well as enh anced resolution; however, the 271/281 fragments could not be resolved using these CE conditions, To investigate the detection sensitivity o f the TAG-stained DNA in capillary electrophoresis, an all-solid-state diode-based, laser-induced fluorescence detector was constructed, whi ch 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, us ing the LIF system, the 271/281 bp fragments were nearly baseline reso lved, with plate numbers exceeding 1 x 10(6) plates/m.