ULTRASENSITIVE NEAR-IR FLUORESCENCE DETECTION FOR CAPILLARY GEL-ELECTROPHORESIS AND DNA-SEQUENCING APPLICATIONS

Electropherograms of oligonucleotides labeled with near-IR fluorescent dyes, separated by capillary gel electrophoresis and detected using an ultrasensitive near-IR fluorescence detection system, are presented. A universal M13 sequencing primer was labeled on the 5' end with a near-IR dye containing an isothiocyanate functional group. Comparison of the on-column detection limits in capillary gel electrophoresis for the near-IR dye-labeled sequencing primer to those obtained for a visible fluorescein-labeled primer indicated improved sensitivity for the near-IR case. The detection limit was found to be 3.4 x 10(-20) mol (SNR = 3) for the near-IR dye-labeled primer, while the on-column detection limit for the fluorescein analog was 1.5 x 10(-18) mol (SNR = 3). The sequence of nucleotide bases in an M13mp18 template was determined using a single lane, single dye technique. The molar concentrations of the ddNTPs used during chain extension reactions were varied to achieve a ratio of 4:2:1:0 (A:C:G:T), which allowed the identification of each terminal base via fluorescence intensity measurements. Sequencing ladders were prepared from the M13mp18 template using standard Sanger dideoxy chain-terminating techniques, the modified T7 DNA polymerase, and the near-IR dye-labeled M13 universal primer. The data indicated reliable sequence determination by the 4:2:1:0 (A:C:G:T) peak height identification method up to 250 bases from the annealing site. Comparison of the known sequence of the M13mp18 plasmid to that obtained using this protocol yielded a base-calling accuracy of 84% for the 4:2:1:0 ratio.