Related Experiment Videos
Fluorescence multiplexing with time-resolved and spectral discrimination using a near-IR detector
Li Zhu1, Wieslaw Stryjewski, Suzanne Lassiter
1Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, USA.
Analytical Chemistry
|August 16, 2003
Summary
This study introduces a novel two-color, time-resolved fluorescence detector for real-time DNA sequencing. The detector achieves high sensitivity and accuracy in capillary gel electrophoresis, enabling longer DNA read lengths.
Area of Science:
- Analytical Chemistry
- Biotechnology
- Spectroscopy
Background:
- Capillary gel electrophoresis (CGE) is a powerful technique for DNA sequencing.
- Real-time fluorescence detection is crucial for efficient CGE data acquisition.
- Existing detectors may have limitations in sensitivity, speed, or spectral resolution.
Purpose of the Study:
- To design and evaluate a novel two-color, time-resolved fluorescence detector.
- To enable simultaneous acquisition of steady-state and time-resolved fluorescence data.
- To improve DNA sequencing fragment analysis during CGE.
Main Methods:
- Development of a detector utilizing two pulsed laser diodes (680 nm and 780 nm).
- Integration of dichroic filters and single-photon avalanche diodes (SPADs) for spectral sorting.
- Real-time data acquisition during capillary gel electrophoresis of DNA sequencing fragments.
Main Results:
- The detector achieved a time response of 450 ps (710 nm) and 510 ps (810 nm).
- Mass detection limits were as low as 7.1 x 10(-21) mol for sequencing primers.
- Demonstrated successful sequencing of an M13mp18 template with a 650 base pair read length at 95.1% accuracy.
Conclusions:
- The developed two-color, time-resolved detector is effective for real-time DNA sequencing analysis.
- The system offers high sensitivity and spectral discrimination, minimizing cross-channel leakage.
- This technology has the potential to enhance throughput and accuracy in DNA sequencing applications.