Color-blind fluorescence detection for four-color DNA sequencing

Abstract

We present an approach called pulsed multiline excitation (PME) for measurements of multicomponent, fluorescence species and demonstrate its application in capillary electrophoresis for DNA sequencing. To fully demonstrate the advantages of PME, a fluorescent dye set has been developed whose absorption maxima span virtually the entire visible spectrum. Unlike emission wavelength-dependent approaches for identifying fluorescent species, the removal of the spectral component in PME confers a number of advantages including higher and normalized signals from all dyes present in the assay, the elimination of spectral cross-talk between dyes, and higher signal collection efficiency. Base-calling is unambiguously determined once dye mobility corrections are made. These advantages translate into significantly enhanced signal quality as illustrated in the primary DNA sequencing data and provide a means for achieving accurate base-calling at lower reagent concentrations.

Comparative microRNA detection from precursor‐microRNA‐transfected hepatocellular carcinoma cells by capillary electrophoresis with dual‐color laser‐induced fluorescence

“…Xue and Yeung used two‐color (488 nm and 514 nm) excitation system for fluorescence detection in DNA sequencing by capillary‐array electrophoresis . Furthermore, Lewis et al also reported the multicolor detection for DNA sequencing based on the pulsed multiline (399 nm, 488 nm, 594 nm, and 685 nm) excitation . In this literature, minimized crosstalk matrix achieved to 0.01 was found in BigDye v3.1‐labeled terminator at 530 nm and 560 nm detection windows.…”

Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR

“…Some methods that have been used to perform color-blind detection in microfluidic systems include pulsed multiline excitation, 22 discrimination by fluorescence lifetimes, 23 dual point excitation, 24 and frequency encoding. 25,26 In frequency encoding, the fluorescence from individual dyes are encoded by pulsing multiple lasers at non-harmonic frequencies of one another and collecting all fluorescence emission onto a single detector.…”

Emerging technologies in DNA sequencing

“…(C) Transformation of the raw trace data derived from the experiment described in B into readable, DNA sequence data using mobility software correction. Reprinted with permission from National Academy of Sciences, U.S.A. © 2005, Lewis et al 2005.…”

“…Recently, Lewis et al (2005) described a simple but effective method for multifluorescence discrimination called pulsed multiline excitation (PME). The underlying principle of this fourlaser system is the correlation of sequential laser pulses with detector response ( Fig.…”

“…In other words, PME measures multicomponent fluorescence assays in a color-blind manner. To demonstrate these advantages, Lewis et al (2005) applied the PME technology to capillary electrophoresis for DNA sequencing. Figure 2B shows the unprocessed signals from the four PME laser waveforms for a portion of the PCR amplicon for the TCF1 (formerly known as HNF1A) exon 10.…”