Laser-induced fluorescence detection by liquid core waveguiding applied to DNA sequencing by capillary electrophoresis.
A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio approximately 250 for peaks around 500 bases. The signal-to-noise ratio is approximately 50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.
Hanning
A
A
Royal Institute of Technology, Department of Analytical Chemistry, Stockholm, Sweden.
Lindberg
P
P
Westberg
J
J
Roeraade
J
J
D004247:DNA / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D007834:Lasers; D009176:Mycoplasma mycoides / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
D004269:DNA, Bacterial; D004247:DNA