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Anal. Chem. 1990, 62,900-903
ARTICLES
High-speed Separations of DNA Sequencing Reactions by Capillary Electrophoresis Howard Drossman, John A. Luckey, Anthony J. Kostichka, Jonathan D’Cunha, and Lloyd M. Smith* Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706
Fluorescently labeied DNA fragments generated in enzymatic sequencing reactions are rapidly separated by capillary gel electrophoresis and detected at attomole levels within the gel-filled caplllary. The application of this technology to automated DNA sequence analysis may permit the development of a second generation automated sequencer capable of efficient and cost-effective sequence analysis on the genomic scale.
INTRODUCTION A central component of the current international effort to map and sequence the three billion bases of DNA encoded within the human genome ( I , 2 ) is the quest for an efficient, high-speed, and cost-effective automated DNA sequencing technology. The current generation of fluorescence-based automated sequencing instruments (refs 3-5; reviewed in ref 6) do not have adequate throughput to permit sequence analysis on this scale at a reasonable cost. If, however, it were possible to increase the throughput of automated sequencers by an order of magnitude or more without a corresponding increase in the cost and complexity of the instrumentation, the cost of sequencing on the large scale required for genomic analysis would approach a reasonable neighborhood. A major limitation to throughput in the automated sequencers is the time required for the electrophoretic separation of the DNA fragments. For example, a typical analysis on an Applied Biosystems (ABI) 370A automated DNA sequencer requires 14 h of electrophoresis to obtain 400 bases of sequence information from each of up to 16 sets of DNA fragments ( 4 ) . If it were possible to significantly decrease the time required for the separation, the capacity of the instrument could be increased correspondingly. Over the past several years capillary electrophoresis (CE) has developed into an analytical technique of considerable power (7-11). In this method electrophoretic separations are performed in very small diameter (typically 50-100 Hm i.d.) capillary tubes. The small dimensions of the capillaries permit the application of extremely high electric fields (as high as 1000 V/cm), and under proper conditions this yields very rapid and high-resolution separations. In unfilled (open tube) capillaries the separation of different species is based primarily upon their different electrophoretic
mobilities in the buffer/solvent system employed. It is also possible to effect separations on the basis of molecular weight, by performing the separations in a gel matrix (8). This approach has been employed for the rapid separation and analysis of synthetic polynucleotides (9),as well as for double-stranded DNA fragments ( I O ) . In this paper we describe the application of capillary gel electrophoresis (CGE) to the analysis of fluorescently labeled DNA fragments generated in enzymatic DNA sequencing reactions. To detect the small amounts of material present, a sensitive laser-based fluorescence detector was constructed with detection limits of about 60 000 molecules of fluorescein. Using this technique it has been possible to reduce the separation time of the DNA fragments by a factor of up to 25 over conventional electrophoresis.
EXPERIMENTAL SECTION CE Instrumentation. Our CE instrument is a breadboard version of the instrument available commerically from Applied Biosystems, Inc. It consists of Plexiglas boxes enclosing two buffer chambers, which can be maintained at constant temperature with a heat control unit. The voltage necessary for electrophoresis is provided by a high-voltage power supply (Gamma High Voltage Research, Ormond Beach, FL) with a magnetic safety interlock, and a control unit to vary the applied potential. Sample injections for open tube capillaries are performed by use of a hand vacuum pump to generate a pressure differential across the capillary (vacuum injection). For gel-filled capillaries, samples are electrophoresed into the tube by application of an electric field (electrokinetic injection). Preparation of Gel-Filled Capillaries. Fifty-centimeter fused silica capillaries (375 wm o.d., 50 Hm id., Polymicro Technologies, Phoenix, AZ) with detector windows (where the polyimide coating has been removed from the capillary) at 25 cm are used in the separations. The inner surface of the capillaries are derivatized with (methyacryloxypropy1)trimethoxysilane (MAPS) (Sigma, St. Louis, MO) to permit covalent attachment of the gel to the capillary wall (12). Briefly, the capillaries are cleaned by successively flowing trifluoroacetic acid, deionized water, and acetone through the column. After the acetone wash, 0.2% solution of MAPS in 50/50 water/ethanol solution is passed through the capillary and left at room temperature for 30 min. The solution is removed by aspiration and the tubes are dried for 30 min under an infrared heat lamp. Gel-filled capillaries are prepared under high pressure by a modification of the procedure described by Bente and Myerson (13). Four percent poly(acry1amide) gels with 5% cross linker
0003-2700/90/0362-0900$02.50/0 C 1990 American Chemical Society
ANALYTICAL CHEMISTRY, V M . 62, NO. 9, MAY 1. 1990
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primer
++
n U
Flgure 1. Laser-induced fluorescence detector for the single wavelength detection of fluorescentiy labeled DNA fragments eiectro
phoresing through a geMiiled capillary column: BPF. band-pass finer: FL, focusing lens: CAP, capillary: CL, coiiection lens: SF. spatlal finer
Time 60.0 - 256.0 min.
(i.e. slits): PMT. photomultiplier tube.
and 8.3 M urea were used for all the studies reported here. A stock solution is made by dissolving 3.8 g of acrylamide, 0.20 g of N,N'-methylenebis(acrylamide),and 50 g of urea into 100 mL of TBE buffer (90 mM Tris borate, pH 8.3,O.Z mM EDTA). Cmss linking is initiated with 10 pL of N,N,N',N'-tetramethylethylenediamine (TEMED) and 250 p L of 10% ammonium persulfate solution. The polymerizing solution is quickly passed into the derivatized column. Filled capillaries are then placed in. 0.d. filled with water, in a steel tube 1m X ' i sin. i.d. X and the pressure is raised to 400 bar by using an HPLC pump and maintained at that pressure overnight. The pressure is gradually released and the capillaries are removed. A short seetion of capillary from each end of the column is removed before use. Separation and Detection of DNA Fragments. Analysis of DNA sequencing reactions separated by conventional electrophoresis was performed on an AB1 370A DNA sequencer. This instrument uses a slab denaturing urea poly(acry1amide) gel 0.4 mm thick with a distance of 26 cm from the sample well to the detection region, prepared according to the manufacturers instructions. The 5'-fluoreseein-labeled oligonucleotide primer used in the sequencing reactions was prepared according to ref 14. The DNA sequencing reactions were prepared as described (15) by using Taq polymerase (Promega Corp., Madison, WI) and were performed on M13mp19 single-stranded DNA template prepared by standard procedures. Sequencing reactions are stored at -20 'C in the dark and heated at 90 "C for 3 min in formamide just prior to sample loading. They are loaded on the 370A with a pipetman according to the manufacturers instructions and on the CE by electrokinetic injection a t 10000 V for 10 s. Laser-Based Fluorescence Detector. Figure 1shows a block diagram of the fluorescence detector used in these experiments. The beam (488 nm, 40 mW) from an argon ion laser (Cyonics, Sunnyvale, CA) is passed through a 488-nm hand-pass filter (Corion Corp., Holliston, MA) and focused with a beam expander (MellesGriot, Rochester, NY) and a 200 mm focal length doublet achromat lens (Melles Griot) onto a 50 pm i.d. fused silica capillary with the beam size adjusted to approximately fill the capillary inside diameter. The emitted fluorescence is collected with a 40X, 0.65 numerical aperture microscope objective (Melles Griot), passed through slits at the objective image plane to eliminate scattered excitation light, and then through a IO-nm hand-pass filter centered at 530 nm (Corion) to select the wavelength region of interest and further reduce scattered excitation light. It is then detected with a Hamamatsu R928 photomultiplier tube, electronically filtered, digitized, and stored in a personal computer for subsequent analysis.
RESULTS AND DISCUSSION Fluorescence Detection. In fluorescence-based automated DNA sequence analysis, detection sensitivity is a critical issue. For example, on the AB1 370A automated DNA sequencer, generally about 0.4 pmol of sequencing reaction (i.e., the DNA fragments enzymatically synthesized f"0.4 pmol of template) is loaded in each lane of the poly(acry1amide)
primer
Time 10.0 - 45.0 min.
Comparison of the separation of fluorescein-labeled DNA sequencing reactions by Conventional slab gel electrophoresis and capillary electrophoresis: (A) conventional electmphwesis on ttm AB1 370A automated DNA sequencer: (6)capillary gel eiectroohoresis of the Same samoie. Conditions are described in the text Flgure 2.
gel. The well in which it is loaded has dimensions of 0.4 mm X 6 mm, or 2.4 mm2. On average, this loading yields about 1-10 fmol of DNA in a given band on a gel. When larger amounts of sample are loaded, the band resolution decreases, presumably because the capacity of the gel has been exceeded. In a 50-pm capillary, the surface area of the top of the gel is 0.002 mm2,or one thousandth of that in the slab gel. This implies that only onethousandth of the sample can be applied without exceeding the gel capacity. Thus one would not expect the hands of DNA detected in a separation by CGE to contain more than about 1-10 am01 of DNA. These considerations suggested from the outset that it would he necessary to have a detection system with excellent sensitivity to detect these small amounts of DNA. Detection limits determined on the system described above by vacuum injection of known volumes of fluorescein labeled oligonucleotide in open (unfilled) tubular capillaries were approximately 0.1 amol (60000 molecules, at a signal to noise ratio of 2 1 ) of injected sample (a 10-nL injection of a lo-'' M solution). Similar detection limits are obtained in the gel capillaries, although the quantification is more difficult due to uncertainties in the efficiency of the electrokinetic sample injection employed (16). Although this system is roughly 10-fold less sensitive than a sheath flow detection system recently described by Dovichi it has the advantage of b e i substantially simpler to use, particularly for applications
(In,
ANALYTICAL CHEMISTRY, VOL. 62, NO. 9, MAY 1, 1990 primer
primer nli I
316.120
I
312.325
I
Time 14.0 - 45.0 min. Figure 3. Separation of fluorescein-labeled T reaction of M13mp19 DNA by capillary electrophoresis. Conditions are described in the text.
such as CGE in which there is no bulk flow of solvent. To evaluate the potential of CGE for DNA sequence analysis, separations of fluorescein-labeled DNA sequencing reactions were performed. Figure 2 shows a comparison of conventional electrophoresis and CGE for the separation of a fluorescein-labeled C reaction of M13mp19 DNA. The conventional electrophoresis employed a 1200-1500 V (30 W, constant power) potential on a 40 cm long, 0.4 mm thick slab gel with a 26 cm distance to the detector from the point of loading (30-37.5 V/cm). The analysis by CGE employed a 10-kV potential on a 50 cm long, 50 Fm i.d. gel-filled capillary with 25 cm to the detector window (200 V/cm). Each plot shows the same region of data, from the primer peak to base 329 (where base 1 is the first base of the primer oligonucleotide; thus the peak designated 329 corresponds to a DNA fragment 329 nucleotides in length), and both data sets were obtained on identical 4% denaturing poly(acry1amide) gels. In this experiment, a 5-fold reduction in separation time in CGE relative to conventional electrophoresis is obtained with somewhat higher resolution for the CGE. On the 6% gels generally used for DNA sequencing in the AB1 370A instrument, the separation time is %fold longer. Thus, these CE data demonstrate a 10-fold decrease in separation time over conventional electrophoresis under standard conditions. Figure 3 shows the separation by CGE of a fluorescein-labeled T sequencing reaction from M13mp19, also performed on a 4% denaturing poly(acry1amide) gel at 200 V/cm. The excellent resolution that may be obtained is evident from an inspection of the data from bases 316-325, corresponding to the sequence TTTTTCTTTT. This clear separation within the T pentet and quartet is better than we generally observe in conventional electrophoresis. In the data presented in Figures 2 and 3, the increased migration velocity observed in CGE is proportional to the increased voltage applied to the gels. Even greater decreases in separation time could be achieved by further increases in the applied voltage. The main limitation to further voltage increases is the stability of the gels. In general, higher fields lead to a more rapid breakdown of the gel structure. This breakdown is characterized by formation of bubbles within the gel and a corresponding drop in current. The reason for this breakdown is not known. At field strengths under 200 V/cm we have been able to use the gels repeatedly over a substantial period of time; a t fields above this, breakdown often occurs fairly rapidly. Figure 4 demonstrates the extremely rapid separations that may be achieved with higher fields. In this experiment the applied field was 400 V f cm, and peaks out to base 213 were
Time 7.5
- 17.5 min.
Figure 4. Separation of fluorescein-labeled G reaction of M13mp19 DNA by capillary electrophoresis using a larger electric field. The applied voltage was 20 kV across the 50 cm capillary tube (400 V/cm). The total time between elution of the primer and the peak 213 bases in length is 10 min.
obtained in 17.5 min from injection of the sample. This is about 25 times more rapid than conventional electrophoresis under standard conditions for the AB1 370A automated DNA sequencer. This observation strongly suggests that if it is possible to develop gel matrices which can withstand larger applied fields, it should be possible to attain further increases in the speed of sequence analysis beyond that which we have described here.
CONCLUSIONS The rapid separations obtained with CGE suggest the possibility of developing a second generation automated DNA sequencer with an order of magnitude or more increased throughput. The high sensitivity of detection could permit a dramatic reduction in the amount of sample required for analysis, perhaps to as little as the DNA obtained from a single M13 plaque (18). Such an instrument would have to be able to analyze many samples concurrently, a t high speed, to be effective. There are at least two ways to approach this problem; by employing many capillaries, run in parallel; or by the use of ultrathin slab gels (12) on which multiple samples may be loaded and analyzed concurrently. In either case, several technical problems will have to be addressed, including developing a detector with sufficient sensitivity and excellent spatial and temporal resolution and developing sample application techniques for efficiently loading such minute samples. If successful, however, the cost effectiveness and throughput of such an instrument would be suitable for the challenges of sequencing on a genomic scale. ACKNOWLEDGMENT We particularly wish to acknowledge the generous support and assistance of Shiaw-Min Chen, Joel Colburn, Paul Grossman, Henk Lauer, and Steve Moring at Applied Biosystems (San Jose, CA), for providing us with CE instruments before they were commerically available, and helping us to rapidly become familiar with their use. LITERATURE CITED Smith, Lloyd M.; Hood, Leroy E. Biotechnology 1987. 5, 933-939. Hood, Leroy E.; Hunkapiller, Michael W.; Smith, Lloyd M. Genomics 1987, 7 , 201-212. Smith, Lloyd M.; Sanders, Jane 2.;Kaiser, Robert J.; Hughes, Peter; Dodd, Christopher; Connell, Charles R.; Heiner, Cheryl; Kent, Stephen B. H.; Hood, Leroy E. Nature 1986, 327,674-679. Conneli, C.; Fung, S.; Heiner, C.; Bridgham, J.; Chakerian, V.; Heron, E.; Jones, B.; Menchen, S.; Mcfdan. W.; Raff, M.; Recknow, M.; Smith, L.; Springer, J.; Woo, S.; Hunkapiiler, M. Biotechniques 1987, 5, (4), 342-348.
Anal. Chem. 1990, 62, 903-909 (5) Prober, James M.; Trainor, (3eorge L.; Dam, Rudy J.; Hobbs, Frank W.; Robertson, Charles W.; Zagursky, Robert J.; Cocuzza. Anthony J.; Jensen, Mark A.; Baumeister, Kirk. Science 1987, 238, 336-341. (6) Smlth, Lloyd M. Am. Biotech. Lab. 1989, 7 (5), 10-25. (7) Jorgenson, James W.; Lukacs, Krynn D. Anal. Chem. 1981, 53, 1298-1302. (8) Hjerten, Stellan J. Chromatogr. 1983, 270, 1-6. (9) Cohen. A. S.: Najarian, D. R.; Paulus, A.; Guttman, A,; Smith, J. A,; Karger, B. L. R o c . Natl. Acad. Sci. U . S . A . 1988, 8 5 , 9660-9663. (10) Compton, S. W.; Brownlee, R. G. Biotechniques 1988, 6 (5), 432-439. (11) Ewing, Andrew G.; Wallingford, Ross A.; Olefirowicz. Teresa M. Anal. Chem. 1989, 61 (4), 292A-303A. (12) Radola, Bertold J. Nectrophoresis 1980, 7 , 43-56. (13) Bente, P. F.; Myerson, J. Hewiett-Packard Company, Eur. Pat. Appl. EP272925, June 29, 1988. US Appl. 946566, Dec 24, 1986.
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Smith, Lloyd, M.; Kaiser, Robert J.; Sanders, Jane 2.; Hood, Leroy E. Meth. in Enz. 1987, 755. 260-301. Applied Biosystems Model 370A DNA Sequencing System I Taq Polymerase Technical Manual; Applied Biosystems: Foster City, CA, 1989. (16) Huang, Xiaohua; Gordon, Manuel J.; Zare, Richard N. Anal. Chem. 1988, 60, 375-377. (17) Cheng, Yung-Fong; Dovichi, Norman J. Science 1988, 242, 562-564. (18) Mead, D., unpublished results.
RECEIVED for review November 6,1989. Accepted February 8,1990. This work was supported in part by grants from the Whitaker Foundation and NIH Grant GM 42366.
Theory for Cyclic Staircase Voltammetry for First-Order Coupled Reactions Mary Margaret Murphy, John J . O’Dea, Dieter A m , and Janet G. Osteryoung* Department of Chemistry, SUNY University at Buffalo, Buffalo, New York 14214
A systematic computational study of three first-order kinetic systems has been performed for cyclic staircase voltammetry. The systems are electrochemically reversible but compikated by a preceding, following, or Catalytic chemical reaction. Theoretical working curves have been Calculated and the variation in peak currents and peak potentials with a change In rate parameter for the chemical step has been compiled in graphical form. The results resemble, but differ quantitatively from, those for square wave and linear scan (cyclic) voltammetry. I t is suggested that reverse current be measured with respect to the zero of current.
A considerable amount of research has already been done by others concerning the application of cyclic staircase voltammetry (SCV) to the study of first-order kinetic systems (1-13). Our purpose here is to explore as fully as possible by computation the usefulness of SCV for determining chemical rate parameters. In particular, we wanted to determine whether rate information for first-order kinetic systems could be gained by simply examining peak currents, peak potentials, peak current ratios, and peak separations and how they vary with the characteristic time of the experiment. Specialists in voltammetric techniques frequently use some type of curve-fitting procedure for analysis of voltammetric data. For the nonspecialist, especially for obtaining estimates of kinetic parameters, and for understanding the broad phenomenological properties of various mechanisms, working curves as presented here are a useful resource. The previous work cited above generally provides only partial information on how peak values can be expected to vary as the time of the experiment is changed. The results presented here are obtained from theoretical voltammograms calculated for the cases where a reversible electrochemical reaction is complicated by a preceding, following, or catalytic chemical reaction. A companion paper deals with slow heterogeneous charge transfer (14). These results are qualitatively or even semiquantitatively the same as results for the analog counterpart of SCV, i.e. linear scan voltammetry (LSV) (15-1 7). (For simplicity we refer to both the unidrectional and cyclic experiment as LSV.) Also, SCV can be viewed as square wave voltammetry with zero square wave amplitude. The relationship between the
net current for a square wave voltammetric experiment (SWV) (18)and the characteristics of the cathodic part of the cyclic staircase curves is easily seen. Although a detailed analysis of the forward and reverse currents in SWV has not been presented, by inspection there is an even closer analogy between the forward and reverse SW and SC currents (18). Cyclic voltammetry is useful for studying reactions with electrochemical or chemical complications because it provides information not only on the initial oxidation or reduction but also on the fate of that initial reduction or oxidation product. The staircase waveform is characterized by two parameters, the step height (AE),and the step width (7). The potential profile in SCV can be described as E = Ei - (j - 1)AE(for E 5 E J and E = E,, + (j - 1)AE (for E I ESP)where E,, is the potential a t which the scan is reversed, (j- 1 )5~t 5 j T and j is the step number (for further discussion see ref 14). The steplike waveform of staircase voltammetry provides the added advantage of discriminating against charging current. This in turn allows faster effective scan rates ( A E / 7 )at a fixed ratio of faradaic to charging current. The stepwise changes in potential are also readily controlled by digital computer. In principle, current can be sampled anywhere along the step (where potential is constant). Experimentally, the upper limit on A E / 7 , and the lower l i i i t on the sampling point along the step, is set by the cell time constant. For the theoretical curves presented below, the current is always sampled at the end of the step. Also, nAE, the normalized step height, is set at a constant value of 5 mV. Therefore the staircase period, 7, is the only variable. Experimentally, it is convenient to specify the time variable in terms of frequency, f = 1 / ~ . One of the criteria for reversibility in LSV experiments is that the peak current ratio (ipa/ipc)be equal to unity. However, determination of the peak current ratio is not always simple since the correct base line from which to measure the reverse (anodic) peak must be determined. First-order EC and CE kinetic systems are treated as the electrode kinetics system described previously (14). Three methods of determining peak current ratios are compared directly in terms of time and applicability. Included here are plots of iw/iw using the extension of the forward current as a base line for reverse peak measurement, a discussion of Nicholson’s semiempirical method for determining iw/iw (19),and a complete set of plots of peak current ratio for which the reverse (anodic) peak is
0003-2700/90/0362-0903$02.50/0 0 1990 American Chemical Society
