James H. Flanagan

Near-infrared, laser-induced fluorescence detection for DNA sequencing applications

Laser-induced fluorescence detection has become the detection strategy of choice in many large-scale DNA sequencing applications due to its ease of Implementation, sensitivity and the ability to identify the constituent bases of DNA in a single separation lane when the probes used have a distinct spectral characteristic. While the common strategy is to use fluorescent dyes which show absorption and emission properties in the visible region (400-600 nm) of the electromagnetic spectrum, our efforts have been directed toward developing near-IR (700-1000 nm) fluorescence as a viable detection strategy for DNA sequencing. In this paper, we discuss our results concerning the use of near-IR fluorescence detection for DNA sequencing carried out in a capillary gel column, where the capillary column has an internal diameter of 75 /spl mu/m, and the loading level of DNA onto this column is in the nL regime, requiring ultra-sensitive detection. In addition, we discuss our efforts toward the development of a highly efficient, single lane, single fluor, base-calling strategy using lifetime discrimination of heavy-atom modified near-IR dyes. The dyes developed for this application contain an intramolecular heavy atom (halogen) on a remote section of the chromophore, resulting in a perturbation in the fluorescence lifetime without altering the absorption or emission maximum of the base chromophore. This will allow the dye series to be excited with a single laser with the fluorescence processed on a single detector and the identity of the terminal base accomplished via lifetime discrimination. In order to effectively carry out lifetime measurements during capillary electrophoretic separation of the oligonucleotides, a simple solid-state time-correlated single photon counting instrument was constructed.

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near‐infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye‐primer versus dye‐terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross‐linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near‐IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye‐terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye‐primer within the gel lane.

Micro-DNA sequence analysis using capillary electrophoresis and near-IR fluorescence detection

Our group is developing optical solid-state, near-IR (NIR) fluorescence detection systems for the analyses of DNA in restriction mapping and sequencing applications. Specifically, we are investigating a base-calling scheme using fluorescence lifetime discrimination in the NIR implementing intramolecular heavy-atom modified NIR fluorescence dyes which can be configured in a single lane, single fluor format. Results are presented concerning the ability to perform lifetime measurements on-line during capillary gel electrophoresis. Due to the high sensitivity associated with NIR fluorescence detection and small injection volumes in capillary electrophoresis, micro-reactor systems are also being developed which will potentially reduce the sample size for preparation of DNA sequencing ladders. The Sanger dideoxy-terminated fragments are prepared using solid-phase sequencing strategies in these micro-capillary reaction chambers. Our discussion focuses on the stability of the immobilized DNA template under typical sequencing conditions and the ability to sequence long templates using this strategy. Finally, we discuss our work on the preparation of nuclear staining dyes which show absorption and emission properties in the NIR for the low-level detection of restriction fragments. Fluorescence spectra of these dyes in the presence of dsDNAs is presented.

Heavy-atom modified near-IR fluorescent dyes for DNA sequencing applications: synthesis and photophysical characterization

A series of near-IR fluorescent dyes have been prepared which contain an intramolecular heavy atom for altering the photophysics to produce a set of probes appropriate for single lane DNA sequencing applications. The identification of the terminal nucleotide base will be affected by temporal discrimination using fluorescence lifetime determination. The heavy-atom modification consists of an intramolecular halogen (mono- or disubstituted) situated on a remote section of the chromophore in order to minimize the perturbation on the photophysics. The series of dyes prepared showed similar absorption and emission maxima as well as fluorescence quantum yields that were similar. However, the lifetimes of these dyes were found to vary with the identity of the halogen substitution, yielding an apparent inverse heavy atom effect, with the heavier atom showing the longest fluorescence lifetime. Nanosecond flash photolysis spectroscopy of these dyes indicated that the intersystem crossing rates in the series increased with the heavier atom, consistent with known heavy-atom effects. The apparent inverse heavy atom effect resulted from decreases in the internal conversion rate of the base chromophore, with the heavier atom showing a smaller rate of internal conversion compared to that of the dye with the lighter heavy-atom modification.

New directions in near-IR fluorescence detection for capillary electrophoresis

Because of the small sample sizes that are typically inserted onto the separation column in capillary electrophoresis (1-100 nL), ultrasensitive detection strategies are required. The common detection approach used in CE is laser-induced fluorescence with He-Cd, Ar or Kr ion laser excitation. We are developing a detector system which utilizes solid-state diode lasers and avalanche photodiodes to produce a low-cost, durable and ultrasensitive fluorescence detector for CE applications. Along these lines, we have prepared some labeling dyes which readily conjugate to primary bioamines and show absorption and emission properties in the near-IR allowing low-level analyses of these target analytes in complex sample matrices. Our discussion will focus on the properties of diode lasers and avalanche diodes for fluorescence detection in CE applications. In addition, we discuss the characteristics of these near-IR dyes and tagging dyes synthesized in our laboratory for the covalent labeling of bioamines and their use in CE. The specific bioanalysis examples that we present utilizing near-IR fluorescence detection for CE are amino acids separations. In addition, we also discuss the ability to do time-resolved fluorescence measurements during CE for peak identification purposes.

Ultrasensitive near-IR fluorescence detection in capillary zone electrophoresis

Capillary zone electrophoresis (CZE) is a powerful new separation technique which possesses the ability to separate small and large molecules. Due to the small volume of material that is typically loaded onto the column, ultrasensitive detection is often required. We have constructed a laser-induced fluorescence detector appropriate for CZE applications using near- IR fluorescence excitation and detection. Separations of native NIR fluorescent dyes in nonaqueous running buffer systems have been performed with detection sensitivities in the low zeptomole range, comparable to state-of-the-art detection limits reported for visible fluorescence detection in CZE. Using our NIR fluorescence detector and NIR labeling dyes synthesized in our laboratory, we present electropherograms of peptides and proteins separated by CZE and detected with NIR fluorescence. We also discuss the basic operating principles of CZE as well as the instrumental components necessary to perform CZE separations.

Single-lane single-fluor sequencing using dideoxy-labeled, heavy-atom-modified near-IR fluorescent dyes

Using a near-IR (NIR) fluorescence detection system and labels synthesized in our laboratories, electropherograms of oligonucleotides separated by capillary gel electrophoresis and detected using NIR fluorescence will be presented. The sequence of nucleotide bases was determined using a single-lane, single-dye technique. The molar concentrations of the ddNTPs used during extension reactions were varied in order to achieve a ratio of 4:2:1:0 (A:C:G:T) which allowed the identification of each terminal base via fluorescence intensity measurements. Sequencing ladders were prepared from the template, M13mp18, using standard Sanger dideoxy chain termination techniques, the modified T7 DNA polymerase, and a NIR-labeled M13 primer. The data indicated reliable sequence determination up to 300 bases with a base-calling accuracy of 90%. In order to eliminate the need for dye-labeled primers and the T7 DNA polymerase enzyme, we have developed a sequencing strategy which utilizes dye-labeled dideoxy nucleotides in a single-lane, single-fluor approach. Base-calling is accomplished by measuring the fluorescence lifetime of intramolecular heavy-atom modified dyes.

Ultrasensitive NIR fluorescence detection and its application to the analysis of DNA

The ability to possess detection sensitivity at the single molecule level is a technically challenging task and will have important applications for the analysis of minute quantities of DNA in applications such as Sanger dideoxy sequencing, restriction maps and scanning confocal microscopy. The ability to detect single visible fluorescent dye molecules in solution has recently been demonstrated. We wish to discuss the first report concerning the detection of single near infrared (NIR) dye molecules in solution using photon burst detection and its application for the analysis of minute quantities of DNA. Near infrared excitation and detection was used to reduce the fluorescent impurity contribution to the background, which temporal and spectral filtering cannot overcome in most cases, allowing sensitive detection in complex biological matrices.