Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection
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- 作者：Ulrike Lieberwirth ; Jutta Arden-Jacob ; Karl H. Drexhage ; Dirk P. Herten ; Ralph Mü ; ller ; Michael Neumann ; Andreas Schulz ; Stefan Siebert ; Gregor Sagner ; Sven Klingel ; Markus Sauer ; and Jü ; rgen
- 刊名：Analytical Chemistry
- 出版年：1998
- 出版时间：November 15, 1998
- 年：1998
- 卷：70
- 期：22
- 页码：4771 - 4779
- 全文大小：229K
- 年卷期：v.70,no.22(November 15, 1998)
- ISSN：1520-6882
文摘
A new one-lane, four-dye DNA sequencing method wasdeveloped which is based on time-resolved detection andidentification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodaminederivatives (MR200-1, JA169), a new oxazine derivative(JA242), and a commercially available cyanine dye (CY5).The dye fluorescence was excited by a pulsed diode laseremitting at 630 nm. The fluorescence decay was detectedby an avalanche photodiode using a single-filter system.The dyes used here, so-called multiplex dyes, can bedistinguished and identified via their fluorescence decaypatterns. The DNA fragments were labeled at the primerusing linkers of various lengths and positions. Forseparation of the enzymatically generated DNA fragments,capillary gel electrophoresis (CGE) with a 5% linearpolyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescencedecay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242),and 1.6 ns (CY5) measured during CGE. The CGEmobility of the labeled DNA fragments could be controlledand nearly equalized by the coupling position and thelinker length. First, time-resolved, one-lane, four-dyeDNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with aprobability of correct classification of >90%. This resultwas obtained directly from the raw data without any ofthe mobility corrections that are necessary with othermethods.