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 a n ultrasensitive near-IR fluorescence detection system, are presented. A universal M13 sequencing primer was labeled on the 5' end with a ne ar-IR dye containing an isothiocyanate functional group. Comparison of the on-column detection limits in capillary gel electrophoresis for t he near-IR dye-labeled sequencing primer to those obtained for a visib le fluorescein-labeled primer indicated improved sensitivity for the n ear-IR case. The detection limit was found to be 3.4 x 10(-20) mol (SN R = 3) for the near-IR dye-labeled primer, while the on-column detecti on limit for the fluorescein analog was 1.5 x 10(-18) mol (SNR = 3). T he 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 achie ve a ratio of 4:2:1:0 (A:C:G:T), which allowed the identification of e ach 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 r eliable sequence determination by the 4:2:1:0 (A:C:G:T) peak height id entification method up to 250 bases from the annealing site. Compariso n 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 r atio.