Technical Notes Anal. Chem. 1995, 67, 1755-1761
Characterization of DNA Size Determination of Small Fragments by Flow Cytometry Jeffrey T. Petty,t Mitchell E. Johnson,tl@Peter M. QOOdwin,t John C. Martin,* James H. Jett,* and Richard A. Keller*st CST-1 and LS-2, MS M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
DNA fragment lengths were determined using the intensity of fluorescent bursts from single fragments stained with a thiazole orange derivative. The individual stained fragments were introduced into a sheathflow cuvette and passed through a low-power (30 mw), continuous-wave laser beam with transit times in the range 3-5 ms. As liffle as 50 fg of DNA was analyzed at a rate of 40 fragments/s for times ranging &om 1 to 15 min. A detectable lower size limit of 1.5 kilobase pairs (kbp) was demonstrated, and a linear relationship between fluorescence intensity and fragment length was observed. Issues relating to size resolution in the 2-50 kbp range are discussed. Many techniques used to analyze DNA involve determination of fragment lengths, which is usually accomplished using electrophoretic migration through a polymeric matrix.' Electrophoretic fractionation of DNA cannot be achieved in free solution because the velocity is independent of size? but the efficiency of electrophoretic sieving of DNA through a polymeric matrix, or gel, does depend on length. Acrylamide and agarose gels have been used to separate fragments of lengths 10-2000 and 10020 000 For larger fragments (>20 000 bp), the electric field strengths are large enough to orient the DNA, which results in loss in size resolution.* To address the problems associated with larger fragments, pulsed-field gel electrophoresis techniques using alternating electric fields were developed, where the time required for reorientation depends on the fragment size.5 Standard laboratory techniques use slab gel electrophoresis? but the desire for faster and more efficient separations, more accurate quantitation, + CST-1.
Ls2. bResent address: Room 308, Mellon Hall of Sciences, Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282. (1) Landegren, U.;Kaiser, R;Caskey, C. T.;Hood, L.Science 1988,242,229237. (2)Olivera, B. M.; Baine, P.; Davidson, N. Biopolymers 1964,2,245-257. (3)Sambrook, J.; Fritsch, E. F.; Maniatis, T.Molecular Cloning, A Loboratoly Manual, 2nd ed.; Cold Spring Harbor Press: Cold Spring Harbor, NY, 1989. Simon, M. N.; Studier, F. W. J Mol. Biol. 1977, 110, (4)McDonell, M. W.; 119-146. (5) (a) Schwartz, D. C.; Cantor, C. R Cell 1984,37,67-75.(b) Cantor, C. R; Smith, C. L.;Mathew, M. IC Annu. Rev. Biophys. Biophys. Chem. 1988,17, 287-304. 4
0003-2700/95/0367-1755$9.00/0 Q 1995 American Chemical Society
higher sensitivity, and automation has led to the development of capillary gel Recently, we9J0and others'l have developed an alternative method of flow cytometric DNA sizing based on measurement of the fluorescence intensity from single fragments stained with a noncovalently bound dye. Fragments of stained doublestranded DNA are passed individually through a focused laser beam to produce a burst of photons from dye fluorescence. When the DNA is stained with a dye, which is not base pair specific, the intensity of the burst is linearly related to the fragment length. The primary advantages of this technique are short data collection times (1-15 min), high sensitivity (as little as 50 fg of DNA), and the ability to analyze larger fragments rapidly. In our plenary work, we demonstrated the technique by sizing DNA fragments in the range 10-48.5 kbp. High background prevented us from detecting smaller fragments with the apparatus as originally configured. There is considerable interest in applying the technique to smaller fragments such as those generated for highresolution physical maps and by polymerase chain amplification techniques. In this work, we characterize the sizing of smaller fragments and describe the changes necessary to accomplish this task. The significant improvements were a redesigned flow system that increased the interaction time of the DNA fragments in the laser beam and carefully optimized spectral filtering to improve the discrimination of the fluorescence signal from the background. With these changes, a 5fold improvement allowed sizing down to 1.5 kbp. It is important to note that further reduction of the lower limit of the size range will require considerable redesign of the apparatus. In addition to extending the size range, these studies confirmed the linear relationship between fluorescence intensity and fragment length for the small fragments. The contributions that limited the resolution in the small fragment region are discussed. (6)Kuhr, W.G.; Monnig, C. A Anal. Chem. 1992,64,389R-407R (7) Clark, S.M.; Mathies, R A Anal. Biochem. 1993,215,163-170. (8)Sudor, J.; Novotny, M. V. Anal. Chem. 1994,66,2446-2450. (9)Goodwin, P.M.; Johnson, M. E.; Martin, J. C.; Ambrose, W. P.; Marrone, B. L.;Jett, J. H.; Keller, R A Nucleic Acids Res. 1993,21, 803-806. (10)Johnson, M. E.;Goodwin, P. M.; Ambrose, W. P.; Martin, J. C.; Marrone, B. L;Jett, J. H.; Keller, R A. Proc. SPIE-Int. SOC.Opt. Eng. 1993,1895, 69-78. (11) Castro, A;Fairfield, F. R; Shera, E. B. Anal. Chem. 1993,65,849-852.
Analytical Chemistry, Vol. 67, No. lo, May 15, 1995 1755
Amplifier
Discriminator
MCS
1
+-I
I I PMT
GPIB
Computer
Fiber
Figure 1. Diagram of the experimental apparatus. See text for details.
EXPERIMENTAL SECTION A diagram of the experimental apparatus is shown in Figure 1. The excitation source was a continuous-waveargon/krypton ion laser (Coherent, Model Innova 70 Spectrum, Palo Alto, CA) operated at 514.5 nm. The beam was coupled into a single mode optical fiber (Newport,Model F-SA,Fountain Valley, CA) using a 20x, NA 0.40 objective (Mol) to provide a high-quality TEMm mode. The output from the fiber was collimated using a lox, NA 0.25 objective (M02) to give a l/e2 beam diameter of 2.9 mm and was focused using a 25 cm focal length lens (L1) to a 46 pm l/e2 beam diameter at the center of a hydrodynamic focusing cuvette (Becton Dickinson, Model 3W1691-000 (Ortho), San Jose, CA). The beam diameters were measured by translating a razor blade through the beam and by fitting the ratio of reflected to incident power to the error function. The radiant power through the cell was approximately 30 mW. At this power, the signal to noise ratio (SNR) was optimal based on the criterion that maximized the signal, which was limited by optical saturation, relative to the background.12 No attempt was made to control the polarization state at the output of the fiber. The fused silica cuvette has a 250 x 250 pm2inner square channel, and its original 4 x 4 mm2 outer dimensions were modifled by grinding and polishing the fluorescence detection side to a thickness of -100 pm to allow placement of the microscope objective at the prescribed working distance. A 40x, NA 0.85 microscope objective with a 390 pm working distance (Nikon, Model CF Fluor 40 x , Garden City, NJ) (M03) was used to collect the fluorescence at 90" from the excitation beam. The collected light was focused onto a 1.2 (horizontal) x 3.0 (vertical) mm2slit (serving as a spatial filter) and passed through a 550 f 15 nm interference filter (Omega Optical, Model 550DF30, Brattleboro,VTj. The transmitted fluorescence was focused by an 8 cm focal length lens onto the photocathode of a thermoelectrically cooled (-30 "C) photomultiplier tube (RCA, Model 31034A, Sommerville,NJ) operated at -1500 V. The photoelectron pulses were amplified, discriminated, and converted to fast NIM pulses (EG & G PAR, Model (12) Peck, IC;Stryer, L.; Glazer, A N.; Mathies, K. A PYOC. Natl. Acad. Sci. U.S.A. 1989,86,4087-4091.
1756 Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
112N Princeton, NJ) that were counted by a multichannel scaler (Stanford Research Systems, Model SR430, Sunnyvale, CA). The multichannel scaler summed the number of pulses in 82 or 164 ps bins; these bin widths were chosen to give 20-60 bins/burst. Data records of 16 384 bins were collected. Each record, correspondingto 1.3-2.7 s of data collection, was transferred via a GPIB interface (National Instruments,Model NBDMA2800, Austin, TX) to a computer (Macintosh, Model IIvx, Cupertino, CA) that used the LabVIEW software package (National Instruments, Version 3.0, Austin, TX) to control the interface. Experiments consisted of 50-300 records. The sample and sheath volumetric flows were controlled by height differences between the sample container, the sheath container, and the drain of the cuvette. The sheath fluid was distilled water (Baxter Healthcare-Burdick and Jackson Div., No. 365-4, high-puritysolvent, Muskegon, MI) flowing at a rate of -10 pL/min corresponding to a velocity of 5 mm/s at the center of the 250 x 250 pm2 cuvette. Longer transit times than used previouslyg were accomplished using a 3 ft section of 280 pm i.d. polyethylene tubing to increase the resistance at the exit of the cuvette. Using a 1 x 10-13 M suspension of 1 pm diameter microspheres (Polysciences, No. 15702,Warrington,PA) delivered at the same volumetric flow rate used for the sizing experiments, the sample stream diameter was measured to be -17 pm by imaging the microsphere fluorescence onto a linear photodiode array (EG & G Reticon, Model RC1000/1001, Princeton, NJ). From this diameter and knowledge of the sheath flow rate, an approximate sample rate of 0.04 ,uL/min was ~alcu1ated.l~ The volume of 10 pL from which fluorescence was detected was determined by the overlap of the sample stream and the laser beam (measured l/e2 diameter = 46 pm). Bacteriophage I. DNA (GIBCO BRL Life Technologies, No. 5250% Gaithersburg, MD) and Hind111 and KpnI digests of I. DNA (New England BioLabs, Nos. 301-2s and 301-7S, Beverly, MA) were stained with the thiazole orange homodimer TOTO-1 (Molecular Probes, No. T-3600, Eugene, OR). The stock DNA solutions were stored at 4 "C, and the TOTO-1 was stored as a 1 mM solution in DMSO at -20 "C. A 1 x M solution of TOTO-1was prepared by diluting 1pL of stock in 99 p L of sterile TE buffer [lo mM Tris-HC1 0. T. Baker, Nos. 4099-02 (Tris) and 953545 (HCl), Phillipsburg,NJ), 1mM EDTA (Sigma, No. E-5134, St. Louis, MO), pH = 81. To stain the DNA, TOTO-1 and then DNA were added to TE to give a total volume of 1000 pL and concentrations of 1.2 x M TOTO-1 and 400 pg/pL DNA These staining concentrationsgive a base pair:dye molecule ratio of 5:l. The resulting solution was incubated for 30-60 min at room temperature (22 "C) in the dark and diluted in TE to give a final total fragment concentration of -1 x 10-13M. The DNA/ dye complex was very stable; narrow burst distributions could be obtained for at least 2-3 h at room temperature. The first step in the analysis of the fluorescence burst data was to determine the transit time of the fragment through the laser beam using the autocorrelation function (Figure 2) .I4 Using the first 16 kB record of burst data from the multichannel scaler, the autocorrelation function was calculated and fit by a half Gaussian. Because the autocorrelation of a Gaussian function yields a Gaussian with a larger width, the standard deviation of (13) Zanin, F.; Dovichi, N. J. Anal. Chem. 1985,57, 2690-2692. (14) Peck, IC;S e e r , L.; Glazer, A N.; Mathies, R A Proc. Natl. Acad. Sci. U S A . 1989,86, 4087-4091.
10x10'
-
!
3.008
0
20
40
60
80
r [bins)
Flgure 2. Determination of the transit time of the fragments through the laser beam from the autocorrelationfunction. The autocorrelation function [A(T)= CE;'d(f) d(t r), where N is the number of data points and d( f ) and d( t t) are the magnitudes of the signal (pe) at times t and t t ] was calculated from the burst data from Figure 3a where WO, and was fit by a half Gaussian [WO WI exp(-(t/w~)~/2), WI, and & are the fit coefficients]. The autocorrelation function is indicated by the open circles, and the fit is indicated by the solid line. The width of the burst is obtained from the fluorescence burst function (FB(t)), which is smaller than the width of the autocorrelation function ( ~ 2 )The . line at 2.1 ms shows the 2a point for the fluorescence burst function, and the transit time is defined as twice this value. The bin width is 82 ps.
+
+
.' 0
t-2
+
0
.B
2
10
4
Bin Number
i 14
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501 40
&
I
+
the autocorrelation fit was divided by (2)112 to give the standard deviation of the fluorescence burst function (um).The transit time of the fragment was d e h e d as b F B , which was chosen so that the transit time corresponds to the l/e2 width of the fluorescence burst from the fragment during the transit of the l/e2 waist of the laser beam. The next stage in the data analysis determined the background by averaging the data below a level set near the maximum of the background noise (Figure 3a). Because the number of points associated with bursts was small (
