Four-color fluorescence detection

Later, a four-color rotary confocal fluorescent scanner was designed to simultaneously detect 96 channels [977] and 384 channels [980]. The confocal scanner used to detect 384 channels on a microcapillary array electrophoresis (pCAE) chip is shown in Figure 7.3 [980],... 

FIGURE 7.3 The rotary confocal fluorescence scanner used to detect pCAE chip separations. Laser excitation at 488 nm (100 mW) is directed up through the hollow shaft of a computer-controlled stepper motor, deflected 1.0 cm off-axis by a rhomb prism, and focused on the electrophoresis lanes by a microscope objective. The stepper motor rotates the rhomb/objective assembly just under the lower surface of the microchip at five revo-lutions/s. Fluorescence is collected along the same path and spectrally, and is spatially filtered before impinging on the four-color confocal detector [980]. Reprinted with permission from the American Chemical Society. 

An automatic sequencing instrument has been developed that uses the chain-terminator method. To avoid the use of radioactive labels, a different color fluorescent dye is attached to the primer in each of the four reactions used to synthesize the DNA fragments. The mixture of fragments from all four reactions is then analyzed using electrophoresis in a single lane. A fluorescent spot appears for each polynucleotide of increasing size. The 3 -terminal base for each spot can be determined by the color of the fluorescence. The detection system is computer controlled, and the acquisition of data is automated. A schematic representation... 

To develop a four-color detectable laser-induced fluorescence (LIF) detection system for capillary elec-... 

Fig. 3 Schematic representation of a 1000-capillary rotary scanner and a four-color confocal fluorescence detection system. (From Ref. [10].)...

Figure 5.5 Fluorescence detection of oligonucleotide fragments produced by the dideoxy method. A sequencing reaction is performed with four chain-terminating dideoxy nucleotides, each labeled with a tag that fluoresces at a different wavelength (e.g., red for T). Each of the four colors represents a different base in a chromatographic trace produced by fluorescence measurements at four wavelengths. [After A. J. F. Griffiths et al., An Introduction to Genetic Analysis, 8th ed. (W. H. Freeman and Company, 2005).]...

There are two methods that have been used to determine fluorescence lifetimes in DNA sequencing. In the first method, known as the frequency-domain or phase-modulation method, the excitation beam is intensity modulated. The a.c. portion of the resulting emission is phase-shifted relative to the laser modulation this phase-shift contains information about the fluorescence lifetime, or lifetimes if more than one fluor is present [140]. McGown and coworkers [144,145] used this method for four-color sequencing. In that work, 488 nm or 514 nm laser light was electronically modulated with a Pockels cell before being focused onto a capillary column. Detection, made normal to the laser direction, was optically filtered to reduce laser scatter and was focused onto the detector of a... 

FIGURE 46.6 The Berkeley rotary confocal fluorescence scanner developed by James R. Scherer. Laser excitation is directed up through the hollow drive shaft of a stepper motor, deflected off axis by a rhomb prism, and is then focused with a microscope objective, tracing a 1-cm radius circular detection path on the chip. Fluorescence is collected along the same path and then spatially and spectrally filtered by a four-color PMT array. The inverted nature of this system allows easy access to the upper surface of a jjiCAF device for electrical, pneumatic, and sample interfacing. 

This entry gives an overview of the fundamentals of DNA sequencing by CAE, jxCAE, and four-color laser-induced fluorescence (LIE) detection, as well as some major factors (sieving matrix, sample preparation, electric field strength, etc.) influencing the sequencing accuracy and efficiency. 

FOUR-COLOR LASER-INDUCED FLUORESCENCE DETECTION AND CAPILLARY ARRAY ELECTROPHORESIS... 

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