Ultrasensitive near-IR fluorescence detection for capillary gel electrophoresis and DNA sequencing applications
| Autor: | Steven A. Soper, Daryl C. Williams |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Detection limit
Gel electrophoresis Chromatography Base Sequence Spectrophotometry Infrared Oligonucleotide Chemistry Molecular Sequence Data Fluorescence spectrometry Analytical chemistry Oligonucleotides Electrophoresis Capillary T7 DNA polymerase Sequence Analysis DNA Fluorescence Analytical Chemistry Electropherogram Capillary electrophoresis Spectrometry Fluorescence |
| Zdroj: | Analytical chemistry. 67(19) |
| ISSN: | 0003-2700 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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