Da Yong Chen

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.

Yoctomole detection limit by laser-induced fluorescence in capillary electrophoresis

Laser-induced fluorescence detection in a sheath flow cuvette is the most sensitive method of detecting fluorescent molecules for capillary electrophoresis. This manuscript demonstrates the detection of 50 yoctomoles (1 yoctomole = 1 ymol = 1 x 10(-24) mol) of rhodamine 6G. A 1-mW He-Ne laser (lambda = 543.5 nm) was used as the excitation source. This detection limit is a 10 times improvement compared to the previous state-of-the-art detection limit in separation science.

Communication. A simple laser-induced fluorescence detector for sulforhodamine 101 in a capillary electrophoresis system: detection limits of 10 yoctomoles or six molecules

High sensitivity fluorescence detection was generated for capillary electrophoresis based on a post-column sheath-flow cuvette. An inexpensive 8 mW He–Ne laser operating at 594 nm was used for excitation of Sulforhodamine 101. This dye, which is commonly used for both protein labelling and deoxyribonucleic acid (DNA) analysis, has molar absorptivity that is matched to the laser wavelength. Collection efficiency was doubled compared with previous work by using two microscope objectives, one located on each side of the illuminated sample stream. A series of injections of Sulforhodamine 101 was performed; the average detection limit (3σ) was 6 ± 1 molecule of dye injected onto the capillary. These injections ranged from 250 to 17 molecules of dye. Both the detection limits and the amount of injection are the lowest ever reported in capillary electrophoresis. The mass sensitivity of this detector is at least two orders of magnitude lower than the best on-column capillary electrophoresis fluorescent detector.

High-efficiency filter fluorometer for capillary electrophoresis and its application to fluorescein thiocarbamyl amino acids

Abstract Fluorescence detection has produced excellent detection limits in capillary electrophoresis. Laser excitation produces the highest sensitivity detection. lie perceived difficulties associated with the use of the laser has discouraged applications of fluorescence to capillary electrophoresis. In particular, difficulties in wavelength selection limit the choice of chemistry available in capillary clectrophoresis. We report the design of a filter fluorometer based on a compact 75 W xenon arc lamp for capillary electrophoresis; the instrument produces detection limits (3σ) of 20 zeptomol (1 zeptomol = 10 −21 mol) for fluorescein and 200 zeptomol for fluorescein labeled amino acids.

Single-color laser-induced fluorescence detection and capillary gel electrophoresis for DNA sequencing

Low zeptomole (1 zmol equals 10-21 mol = 600 molecules) detection limits are produced for DNA sequencing by capillary gel electrophoresis. A 750 (mu) w green helium-neon laser ((lambda) equals 543.5 nm) is used to excite tetramethylrhodamine-labeled DNA fragments in a sheath-flow cuvette. A cooled photomultiplier tube is used to detect fluorescence in a single spectral channel. Sequencing data is generated at a rate of about 70 bases/hour.