Anal. Chem. 2006, 78, 3632-3637
Solid-Support Sample Loading for DNA Sequencing Jo 1 rn Ueberfeld, Sameh A. El-Difrawy, Korisha Ramdhanie, and Daniel J. Ehrlich
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142
We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel. This method circumvents both the minimum volume requirement of pipettors (since only solid beads are transported) and the timing complications of double-T microfluidic injection. Injections from Sanger samples of 10-times the starting DNA sample. Sequencing traces show a resolution that matches or exceeds double-T injections. A kinetic model reproduces the time-dependence of the injection signals and proves that total nonidealities in the method produce injection-broadened plugs of ∼1-s duration. The method should be broadly extendable to DNA and protein separations in both microdevice and capillary electrophoresis. Current large-scale DNA sequencing efforts are largely based on the enzymatic chain-termination method developed by Sanger et al.1 The resulting DNA fragments are separated using capillary array electrophoresis (CAE) machines. Laboratory robotic work stations used in conjunction with these sequencers have minimum working volumes of ∼1 µL, which is an important limitation in lowering sequencing cost at both the sample preparation and the electrophoretic read-out stages.2 Microfabricated capillary arrays have the potential for a nearterm cost reduction relative to CAE machines due to their increased channel density and parallelism.3-6 These advantages should reduce the capital cost at genome centers and shift effort onto scaling disposable costs. Whereas the throughput is significantly enhanced as compared to the CAE machines, there are (1) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463-5467. (2) Jovanovich, S. B.; Salas-Solano, O.; Li, J.-t. In PCT Int. Appl.; Amersham Biosciences (SV) Corp.: Piscataway, NJ, 2003; p 183. (3) Aborn, J. H.; El-Difrawy, S. A.; Novotny, M.; Gismondi, E. A.; Lam, R.; Matsudaira, P.; McKenna, B. K.; O’Neil, T.; Streechon, P.; Ehrlich, D. J. Lab Chip 2005, 5, 669-674. (4) El-Difrawy, S. A.; Lam, R.; Aborn, J. H.; Novotny, M.; Gismondi, E. A.; Matsudaira, P.; McKenna, B. K.; O’Neil, T.; Streechon, P.; Ehrlich, D. J. Rev. Sci. Instrum. 2005, 76, 074301/074301-074301/074307. (5) Paegel, B. M.; Emrich, C. A.; Wedemayer, G. J.; Scherer, J. R.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 574-579. (6) Liu, S.; Ren, H.; Gao, Q.; Roach, D. J.; Loder, R. T., Jr.; Armstrong, T. M.; Mao, Q.; Blaga, I.; Barker, D. L.; Jovanovich, S. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5369-5374.
3632 Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
further opportunities to reduce sample requirements; however, these front-end scaling opportunities have been largely unexplored. For example, in our laboratory most sequencing data (nearly 100 000 lanes of data3) were taken at minimum practical quantities of 7.5-18.7 ng of sample (2-5 µL of a “1/32 x” BigDye terminator solution per channel; see definition of sequencing concentrations in the Materials and Methods Section). This matches, but does not exceed, the standard operating samples used in many large genome centers.7 Another obstacle for sample scaling is the generation and purification of the Sanger sequencing reaction. Ethanol precipitation remains the major method for cleanup, although it is laborand time-consuming. An alternative method, the solid-phase reversible immobilization (SPRI), developed by Hawkins et al.,8-10 is based on the nonspecific adsorption of DNA onto micrometersized paramagnetic beads. In this case, the DNA is adsorbed to carboxyl-modified microspheres in the presence of divalent cations; however, the method would be extendable to many other analytes by alternative derivatizations. Because the microspheres are paramagnetic, they can be collected out of suspension by applying an external magnetic field. A wash step removes the unincorporated nucleotides, enzymes, and excess salt, but not the extension products. The latter are selectively eluted with pure water. Additionally, there is some impetus to develop direct injection methods for microdevices. The double-T injector is the overwhelming choice for generation of small plugs in all microdevice electrophoresis, including our own work. However, it was shown that one of the largest challenges in maintaining consistent longterm performance in microdevice sequencing arrays stems from the tight “timing window” associated with T-injection.3 Relatively small channel-to-channel variation in resistivity and leakage currents leads to uncontrolled variation across the array in optimum timing for switching from sample-to-waste (load) to cathode-to-anode (run) voltage (Figure 1). This optimum timing window is often measured in tens of seconds. Although the various cross-channel injection methods of microdevices are elegant, direct injection, if it can be implemented, may have the advantage of greater array stability and reproducibility.11 Here, we present a new method for loading small DNA samples onto microfabricated DNA sequencing devices. We use a modified (7) Grills, G. (Harvard Partners Genome Center, Boston, MA); Cahill, P. (Broad Institute, Cambridge, MA); personal communication. (8) Elkin, C. J.; Richardson, P. M.; Fourcade, H. M.; Hammon, N. M.; Pollard, M. J.; Predki, P. F.; Glavina, T.; Hawkins, T. L. Genome Res. 2001, 11, 1269-1274. (9) Elkin, C.; Kapur, H.; Smith, T.; Humphries, D.; Pollard, M.; Hammon, N.; Hawkins, T. Biotechniques 2002, 32, 1296-1302. (10) Hawkins, T. L.; Oconnormorin, T.; Roy, A.; Santillan, C. Nucleic Acids Res. 1994, 22, 4543-4544. 10.1021/ac052201x CCC: $33.50
© 2006 American Chemical Society Published on Web 05/02/2006
Figure 1. The “timing window” problem for high-throughput microdevice sequencing. In large micromachined arrays that rely on double-T injection, uncontrolled resistances (Ri) and parasitic currents (Ii) cause uncontrolled channel-to-channel variations in the timing for DNA loading.1 This causes unpredictable variations in optimum timing for switching between loading voltage [from sample (Si) to waste (Wi)] and run voltage [from cathode (C) to anode (A)].
and highly miniaturized version of SPRI for the cleanup of DNA sequencing reactions and the direct injection into microchips. This method, through sample scaling, may be the basis for sequencing cost reductions and, through direct electrokinetic injection, may produce a large advantage in reproducibility and optimization of DNA sequencing on microfabricated arrays. MATERIALS AND METHODS Chip Preparation. Electrophoretic microdevices were fabricated from 1.1-mm-thick alumina silicate glass (Corning 1730, Corning Co., Corning NY) using techniques described previously.12,13 The separation channel was 11.5 cm in length, and the two side channels had a length of 2.5 mm each. The structure was 40 µm deep and 90 µm wide at the top. The side channels forming the injector were offset lengthwise by 250 µm, forming a double-T. Glass reservoirs (Ace Glass, Vineland, NJ) of 50-µL volume were affixed around the channel access holes to hold sample and buffer using optical adhesive according to the manufacturer’s specification (Nordland Products, New Brunswick, NJ). Channel surfaces were coated with linear polyacrylamide (LPA) using a modified Hjerten procedure.12,14 Electrode Preparation. One end of a permalloy-80 wire (0.5mm diameter, 5-cm length, ESPI International, Ashland, OR) was ground to the desired shape using lapping paper. Scanning Electron Microscopy. Electrode tips carrying magnetic microspheres were coated with Au using a sputter coater. Imaging was carried out with a JEOL JSM-5600LV scanning electron microscope. Separation Matrix Preparation. The LPA separation matrix was purchased from Dakota Biosciences, LLC (Sioux Falls, SD). LPA solutions (4%) were prepared with 1× TTE (50 mM Tris/50 mM TAPS/2 mM EDTA) and 7 M urea. The solutions were ready for use after 3 days of slow stirring in a glass jar. DNA Sample Preparation. Samples were prepared with the Applied Biosystems (ABI) Big Dye Terminator 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) using the Geneamp (11) Srivastava, A. Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, MA, 2002. (12) Koutny, L.; Schmalzing, D.; Salas-Solano, O.; El-Difrawy, S.; Adourian, A.; Buonocore, S.; Abbey, K.; McEwan, P.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 2000, 72, 3388-3391. (13) Schmalzing, D.; Adourian, A.; Koutny, L.; Ziaugra, L.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 1998, 70, 2303-2310. (14) Hjerten, S. J. Chromatogr. 1985, 347, 191-198.
PCR System 9700 with 20-µL reactions. One reaction contained 1 µL of Big Dye Terminator reaction mix, 3.5 µL of 5× ABI Sequencing Buffer, 3.2 pmol of M13 forward primer (Gene Link, Inc. Hawthorne, NY), and 100 ng of M13 mp18 template (New England BioLabs, Beverly, MA). Cross-Correlation of Sequencing Sample Quantities. The vendor of the terminator-ready reaction mix (Applied Biosystems, Foster City, CA) recommends the use of 8 µL of terminator-ready reaction mix per 20 µL cycle sequencing reaction. Our laboratory routinely uses 1 µL of the reaction mix in a 20-µL reaction, which in the parlance of the genome centers, corresponds to a “1/8× Sanger chemistry”. The concentration of the purified extension products (ethanol precipitation) redissolved in 20 µL water is ∼15 ng/µL, as determined by absorption measurements at 260 nm. For routine sequencing runs on our T-injected microchips, we use 6 µL of a 4-fold diluted purified sample, e.g., 6 µL of a “1/32× reaction”. This corresponds to a total amount of 22.5 ng of DNA. Large genome centers typically load as little as 2-µL of a “1/64× reaction” on one capillary of a production CAE machine.7 Assuming the same reaction and purification efficiency, this would correspond to 3.75 ng total of DNA per run. DNA Adsorption to Paramagnetic Microspheres. One microliter of a paramagnetic bead suspension (1-µm bead diameter, COOH modified surface, 5% w/w, Seradyn, Indianapolis, IN) was washed three times with 50 µL of MilliQ purified water and resuspended in 20 µL of water. One microliter of this suspension contains ∼3.5 million beads, as can be calculated from the bead density of 1.36 g cm-3 (given by the manufacturer) and the bead diameter of 1 µm. One microliter of the bead suspension, 4 µL of 1× sequencing buffer (Applied Biosystems, Foster City, CA), and 1 µL of 1/8× Sanger sequencing reaction (see below) were mixed in a 200-µL Eppendorf tube. After addition of 50 µL of 100% ethanol (Aaper Alcohol and Chemical Co., Shelbyville, KY), the suspension was shaken for 15 min. The beads were captured at the tube wall with permanent magnets (diameter 6 mm, thickness 3 mm, Digikey Corporation, Thief River Falls, MN), and the supernatant was removed. After washing with 20 µL of 70% ethanol, the beads were resuspended in 10 µL of 70% ethanol and divided into five aliquots. For studies of the capacity for DNA uptake, the beads were resuspended in water and shaken for 5 min. UV absorption was measured with a Nanodrop device (Nanodrop Technologies, Wilmington, DE) after removing the beads. Preelectrophoresis, Injection, and Run Conditions. Between runs, the separation matrix was replaced. The gel was preconditioned by applying 190 V/cm between the anode and cathode and 350 V/cm between the waste and sample until the current reached a plateau. Beads were captured at the electrode tip by fixing several permanent magnets (diameter 6 mm, thickness 3 mm, Digikey Corporation, Thief River Falls, MN) at a distance of 5 cm from the polished end of the electrode and dipping the electrode end into the bead suspension. Quantitative extraction of the beads from the suspension was observed with a stereomicroscope. The permalloy-80 electrode carrying the magnetic beads was positioned over the sample injection port by means of a micromanipulator, carefully avoiding contact between the tip and water. The final distance between tip and port was 1 mm. Then the desired voltage was applied between the sample and anode port, and finally, 20 µL of water was added to complete the loading circuit. After release, DNA was driven into the Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
3633
Figure 2. DNA uptake on paramagnetic microspheres. Recovered DNA is plotted against the volume of “1/8×” Sanger sequencing reaction that was used for the extraction. (For definition of 1/8× concentration, see text.)
separation channel for 60 s by applying 190 V/cm sample to the anode, the permalloy electrode was removed from the sample well, and the electrophoresis was continued between cathode and anode ports. Both reservoirs were filled with 1× TTE buffer. The run voltage was 190 V/cm. Fluorescence was excited with an Ar-ion laser at a distance of 4 mm from the sample port (for injection profile determination) or 115 mm after the sample port (for full sequencing traces). Data Reduction. Resolution curves for the sequencing electropherograms after 11.5 cm were calculated from the G channel. Resolution is defined as
R ) x[2 ln 2] (xB2 - xB1)/((wB2 + wB1)(B2 - B1)) where B1 and B2 are subsequent bases located at a migration time of xB1 and xB2, respectively, with measured full widths at half maximum (fwhm) of wB1 and wB2, respectively. RESULTS Bead Characterization. In an initial experiment, the loading capacity of the paramagnetic microspheres was tested. For this purpose, the beads were incubated with different volumes of unpurified Sanger sequencing reaction mix, washed, and resuspended in water to elute the DNA. The final DNA concentration was determined with UV absorption measurements. The DNA uptake was linear up to a reaction volume of 10 µL (Figure 2). With increasing volumes of sample solution, the surface of the microspheres becomes saturated, and the DNA uptake reaches a plateau of ∼7.5 pg per 1-µm-diameter bead. In our injection experiments, we typically adsorbed 0.1-1.0 ng total of DNA out of solution onto 3.5 million beads, which corresponds to 29-290 ag DNA per bead. Direct Electrokinetic Injection and Electrode Shape. To directly inject the DNA from the paramagnetic beads into a microchannel, the original SPRI protocol requires modification. Figure 3 represents the steps of our “miniaturized” protocol. The first two steps (incubation of the beads with Sanger sequencing reaction and removal of unincorporated dideoxynucleotides) are similar to the previous procedure.9 Then the beads are resuspended in 70% ethanol and captured with a magnetized permalloy wire. Next, the bead-coated wire (which is to serve as an electrode) is positioned over the sample port by means of a micromanipulator. Care must be taken not to immerse the electrode into water, because this would elute the DNA from the 3634 Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
Figure 3. Miniaturized solid-support loading protocol for the direct injection of DNA into microchannels. This is a modification of the SPRI method. Paramagnetic beads are added to scavenge extension product. The beads themselves are then captured on a magnetized wire and are transported to the microchip. The wire is then used, in a one-step process, as an electrode for direct electrokinetic injection.
beads and result in a dilution of the concentrated sample. For injection, high voltage (0.1-1 kV/cm) is applied to the electrode, then water is added with a pipet to complete the electrical circuit and to simultaneously elute the DNA from the captured beads. In some experiments, a short high-voltage pulse, termed “release voltage”, is applied immediately after the circuit is completed. The initial experiments used simple (flat-ended), cylindrically shaped permalloy and stainless steel electrodes of various metallurgical compositions; however, injection profiles obtained with this electrode at run voltage 190 V/cm were irreproducible and often reflected multiple peaks (Figure 4a). Electrode Geometry. The multipulsing phenomenon shown in Figure 4a was studied with video microscopy and found to result from sporadic release of dense associations of paramagnetic beads. Both the electric and magnetic field distributions from the electrode tip geometry play a role in these phenomena. In the first stage, since the force on individual beads is proportional to the product of the field (B) and magnetic flux divergence (∇B),15,16 the magnetic field draws beads to regions of high magnetic flux divergence∇B. For near-cylindrical geometries, this causes bead accumulations at the cylinder “corners”. Moreover, the cooperative attraction of the paramagnetic particles causes beads to plate around on all sides of the “corners” (Figure 5a). When the electric field is applied, some of these plated bead assemblies are in regions of lower electric field, and these “clumps” come off the tip at later times, leading to multipulsing of the DNA injection. In the experiment, multipulsing was reduced but not eliminated by driving the release with short high-value voltage pulses (e.g., 2.6 kV/cm for 600 ms). A better solution was found by modifying the tip geometry (rounding it) to eliminate the high-curvature “corners”. This change eliminates the regions of high ∇B and produces a uniform thin coverage of the paramagnetic beads on the electrode tip (Figure 5b). The uniform coating is released in a single injection pulse, even at modest voltage (Figure 4b). (15) Knoepfel, H. E. Magnetic Fields; Wiley & Sons: New York, 2000. (16) Jackson, J. Classical Electrodynamics; Wiley & Sons: New York, 1975.
Figure 6. Injection plug widths (fwhm in seconds at 4-mm observation distance) for increasing release voltage for the two electrode geometries investigated (0, cylindrical (flat-ended) tip; 2, rounded tip). The points at the extreme left are obtained at a low, 8.4V/cm, release voltage. Results were averaged over multiple runs, and multipulsing is treated as the fwhm for all peaks at >50% of the maximum amplitude.
Figure 4. Injection profile obtained with direct electrokinetic injection from (a) a flat-ended cylindrical electrode and (b) a rounded electrode. The G-channel signal from a Sanger-sequencing sample prepared from M13 template is shown at an observation distance of 4 mm into the matrix-filled separation channel. Observation of the flat-ended cylindrical electrode with a microscope during the injection process revealed the release of clumps of beads at time intervals similar to the peak separation in the electropherograms. The injection from the rounded electrode (b) shows a single, well-formed sample plug.
Figure 7. On-chip calibration of directly injected DNA using the solid-support method. G-channel readings. The injected signal strength for optimized double-T injection (250-µm injector length) using 6 µL of 1/32× Sanger samples (but 22.5 ng DNA, i.e., >50times more DNA loaded on chip) is shown for comparison.
Figure 5. Scanning electron micrographs of (a) a cylindrical (flatended) and (b) a rounded magnetized permalloy-80 electrode carrying paramagnetic microspheres (250× magnification). In (a), the beads are drawn by the high divergence of the magnetic field at the “corners” of the cylinder, where they accumulate in dense assemblies bound by paramagnetic attraction. In (b), the high-curvature “corner” of the electrode has been eliminated. Note the uniform coverage of paramagnetic beads formed by the more uniform magnetic field divergence from this tip.
The behavior of both cylindrical and rounded electrodes is quantitatively illustrated in Figure 6, where the injection peak widths are shown as a function of the strength of the initial voltage pulse. The electrode with a flat cylinder tip showed a longer
plug (or multiple peaks) at low field strength. The peak widths obtained with the rounded tip remain narrow over the whole voltage range. On-Chip Calibration Curve. Having established a reliable injection protocol, we verified its validity over the concentration range of interest. For this purpose, we established a calibration curve from 50 pg to 1 ng total starting DNA, initially suspended in reactions of 5-µL volume. The graph shows a linear relationship between DNA concentration and peak area (Figure 7). It is worth mentioning that a 2.0 OD filter was used in these measurements to reduce the intensity of the laser light. By omitting the filter, femtogram amounts of DNA should easily be detectable. DNA Sequencing. In the next step, we recorded the fluorescence intensity at a distance of 11.5 cm from the sample port to check if our injection protocol is suitable for DNA sequencing. We injected from the microbeads using the low-voltage roundedtip protocol then drove samples from the sample arm into the separation channel for 60 s. Then the voltage was applied between the cathode and anode reservoir until the run was completed. Sequencing traces showed good peak-to-peak resolution for extension products between 150 and 600 bp, as was characteristic of the particular LPA sieving matrix and the channel length. A Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
3635
Figure 8. G-channel traces for short-chip (11.5 cm) sequencing using double-T, 22.5 ng DNA loaded on chip (a) and solid-support sample injection, 1 ng DNA loaded on chip (b). Sequencing is at 50 °C and conditions similar to refs 12 and 13.
reduction in the LPA concentration to ∼2% and an increase in channel length to ∼40 cm should result in performance near the state of the art for long-read sequencing.12 Figure 8 shows a direct comparison between solid-support injection and a conventional double-T injection of the same sample. It is known that sample stacking at both the sample well/matrix interface and inside the double-T injector17-21 aid in the narrowing of the double-T injection plug. Nonetheless, the solid-support injection shows a narrower plug over a very appreciable region of the resolution curve (Figure 9). This is reflected in significantly higher resolution values for the central section of the sequencing trace. This is consistent with the result expected on the basis of the several-second-wide injection plugs seen (after less sieving) at the 4-mm detection distance (Figure 4b). Kinetic Model. We have constructed a simple 1-D model to deconvolve the contributing factors of the new sample-injection kinetics (DNA desorption and bead kinetics) from electrophoresis in the sample well and matrix-filled channel in the presence of time-dependent local fields. Even in the simplest case, the 1-D field redistributes rapidly at the buffer-matrix interfaces19-21 and distorts the fragment distribution in the injection pulse. Therefore, numerical modeling of this kind is necessary to distinguish the new injection-related effects from the plug distortion at the matrix interface and subsequent sieving before the plug reaches the observation point used in the experiment. The loading channel is represented as a 1-D array 120 mm in length between cathode and anode. The first 1 mm at the cathode end is the buffer-filled length in the sample well, followed by 118.5 (17) Srivastava, A.; Metaxas, A. C.; So, P.; Matsudaira, P.; Ehrlich, D.; Georghiou, G. E. Electrophoresis 2005, 26, 1130-1143. (18) Vazquez, M.; McKinley, G.; Mitnik, L.; Desmarais, S.; Matsudaira, P.; Ehrlich, D. Anal. Chem.; 2002, 74, 1952-1961. (19) Spencer, M.; Kirk, J. M. Electrophoresis 1983, 4, 46-52. (20) Spencer, M. Electrophoresis 1983, 4, 36-41. (21) Bilenko, O.; Gavrilov, D.; Gorbovitski, B.; Gorfinkel, V.; Gouzman, M.; Gudkov, G.; Khozikov, V.; Khozikov, O.; Kosobokova, O.; Lifshitz, N.; Luryi, S.; Stepoukhovitch, A.; Tcherevishinick, M.; Tyshko, G. Electrophoresis 2003, 24, 1176-1183.
3636
Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
Figure 9. Resolution curves for sequencing of M13 template, 11.5cm chips using double-T (250-µm) (9) and solid-support injections (O). The solid-support injection achieves higher resolution between 150- and 575-bp length. The solid lines are second-degree fits to the resolution values (bold line, solid-support injection; thin line, double-T injection).
mm of matrix-filled channel and 0.5 mm of buffer at the anode well. Major ion carriers in the channel are TAPS, Tris, and chloride. The model of local species concentration change with time under the different forces is represented as
∂Ci ∂Γ + )0 ∂t ∂x
(1)
where Ci is the concentration of the ith species in moles/m3, and Γ is the flux in moles/(m2•sec). The total flux consists of two components, Γ ) Γa + Γd, where the flux due to advection is Γa ) vici,vi ) µiziE, and where zi is the signed valance of the specific species, and µi is its mobility. The flux due to diffusion (Γd) is calculated as Γd ) - Di ∂Ci/∂x, where Di is the diffusion coefficient. We can rewrite eq (1) as
(
)
∂Ci ∂Ci ∂ + + µiziECi ) 0 -Di ∂t ∂x ∂x
(2)
The differential eq (2) is rewritten in a discrete form as
Ci(t + ∆t, x) ) Ci(t, x) +
Di∆t
(Ci(t, x + ∆x) - 2Ci(t, x) + (∆x)2 ∆t (C (t, x + ∆x)vi(t, x + ∆x) Ci(t, x - ∆x)) 2∆x i Ci(t, x - ∆x)vi(t, x - ∆x)) (3)
We model the sample well as a constant volume reservoir with an initial uniform DNA concentration. A typical loop to simulate the system consists of the following steps: •calculate local concentration using eq (3),
Table 1. Measured and Predicted fwhm’s and Inferred Release-Related Width for M13 Injection Plugs at the 4-mm Detector Location for Two Extreme Values of the Release Voltage (600-ms Release Pulses) release voltage
peak height (V)
1250 V/cm 8.4 V/cm
0.9 1.2
fwhm (s) measured predicted 2.0 1.8
1.6 1.6
inferred total release-related widtha (s) 1.1 0.72
a Determined by assuming a sum of squares model and includes all nonideal factors related to the solid-support injection.
Figure 10. Simulation results (thin line) in comparison to the measured profiles (bold line) for M13 Sanger-sequencing samples at different voltage for the 600-ms release pulse at a detector distance of 4 mm into the matrix-filled separation channel. Direct injection and instantaneous δ-function release of DNA into the 1-D column are assumed in the simulation. The width of the loading profile did not change significantly with the release voltage. The falloff of the simulated profile is faster than that of the actual profile and reflects the deviation of the actual result from the case of δ-function (instantaneous) release.
in the matrix were taken at 10 and 20 mM, respectively. The concentration of the same ions inside the sample well was modeled using a 1/2 Gaussian, with changing variance (80-120) and mean point at the water/matrix interface. In both cases, the simulations showed changes of
