Time-resolved DNA identification in capillary gel electrophoresis with semiconductor lasers

摘要

We developed a simple, tiny setup with only a few optical devices for fast and sensitive identification of DNA with a short-pulse semiconductor laser operating at 20 MHz. In combination with newly synthesized fluorescent dyes (rhodamine derivatives) which exhibit high fluorescence quantum efficiencies and distinct fluorescence lifetimes at semiconductor laser excitation wavelength a sensitive detection and time-resolved identification of DNA can be achieved. At an excitation wavelength of 635 nm the fluorescence background is greatly reduced. We demonstrate the DNA identification of A- and G-terminated DNA fragments labeled at the 5'- end with the rhodamine derivatives MR 200-1 and JA 169 during capillary gel electrophoresis (CGE). The characteristic time-resolved data are acquired by the time-correlated single-photon counting (TCSPC) technique. Time-resolved identification analysis is realized by the maximum likelihood estimator (MLE). For prediction of the error rate (misclassification) Cramers equation in combination with a pattern recognition technique is applied. These methods deliver high reliabilities at low classification error rates for low fluorescence light level applications.