Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection

Abstract

A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of > 90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods.

Stains, labels and detection strategies for nucleic acids assays

“…44 An important characteristic of the detection dye in this context is that the excitation maximum of the dye^nucleic acid complex should be close to an available laser wavelength. Stains used in CE include SYBR 1 Oxazine derivative JA242 80 Platinum coproporphyrin 81,82 Rhodamine derivatives MR200-1 and JA169 80 5-Carboxyrhodamine (RHD) 83 Rhodamine green 29 Carboxy-X-rhodamine (ROX) 59,75 Carboxytetramethylrhodamine (TAMRA) 59 Tetrachloro-6-carboxy uorescein (TET) 83 Texas Red …”

<title>Analysis of ultrasensitive fluorescence experiments</title>

“…1 For example, for 'conventional' DNA sequencing by the Sanger method, the electrophoresis within a capillary or slab gel may be multiplexed by the use of spectroscopically identifiable fluorescent labels. Lieberwirth et al 2 have recently demonstrated such multiplex dye DNA sequencing in capillary gel electrophoresis by the use of four dyes with disparate fluorescence lifetimes. The best algorithm for classification was found to be "a special pattern recognition technique" , which is equivalent to the maximum likelihood estimation (MLE) technique3 described in section 3.…”

“…To compare P(m3 A) and P(m3 fB) we need only compare the ratio P(mjIA)/P(mIB) = H(aj//3j)mi (2) with 1, or the logarithm of the ratio ln[P(mIA)/P(mIB)] = mln(a//3) (3) with 0.…”

Capillary Electrophoresis of Single-stranded DNA

“…Among several modes of separation, CGE has been extensively employed since the gel matrix provides the capability of sieving for different sizes of DNA fragments. 15 CGE has been extensively employed for the study of DNA sequencing, 16 DNA restriction fragment analysis, 17 and polymerase chain reaction (PCR) fragment analysis. 18 In CGE, one-or four-base resolution was easily obtained by the proper choice of gel matrix and concentration.…”