Anal. Chem. 1982, 64, 1967-1972
1987
Noninvasive Imaging of Nanogram Quantities of DNA in Agarose Electrophoresis Gels Maureen Lanan, Daniel W. Grossmann, and Michael D. Morris' Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055
Image procerring techniques which separate the DNA Kerr effect and Induced electroklnetlc dlstortlon contrlbutlons to ekctrlc blrefrlngence Images of agarose nuclelc acld electrophoresisgels are declcrlbed. Under standard electrophoresk condltlons, detection Ilmltr of 10 ng of DNA per well are obtalned In hydroxyethylatedagarosewlthout slgnal averaging or 7.5 ng wlth averaglng of four measurements. Malntalnlng constant gel temperature Is shown to Improve the quallty of the Images. Monochromatlc llght (589 nm) Is shown to give a small Increase In sensltlvlty compared to polychromatlc (650 f 20 nm) Ilght.
INTRODUCT10N Nucleic acid fragments in agarose electrophoresis gels are most commonlyvisualized by staining with ethidium bromide and visualizing the fluorescenceunder UV illumination. While simple and sensitive, ethidium bromide fluorescence is not ideal in two important applications of agarose gel electrophoresis: preparation of large DNA fragments and on-line monitoring of DNA fragments longer than approximately 100 kilobase pairs (kbp). Electric birefringence (EBI) provides a promising alternative to existing dye and/or UVbased optical detection methods. In this communication, improvements in EBI signal processing are presented along with a brief investigation of analytical parameters important in EBI. Large DNA fragments are sensitive to damage (nicking) by UV light. Because a major purpose of gel electrophoresis is isolation of DNA fragments for recovery and use in other experiments, it is important to develop visualization procedures which avoid nicking and which are as simple and rapid as possible. The nicking problem is sufficiently serious that a common preparative procedure is to separate portions of a nucleic acid fragment mixture in two adjacent lanes,4 one of which is stained after physically cutting it out from the gel. After visualization of the stained lane, corresponding regions of the unstained lane are excised. Because the lane alignment is not perfect, the recovered DNA must be subjected to a second electrophoresis to check its purity. Stainless, nondamaging detection would speed and simplify such preparative procedures. On-line detection of large DNA fragments during pulsedfield gel electrophoresis requires development of a stainless detection method. Pulsed-field electrophoresis of large fragments requires times which may reach or exceed 24 h. The ability to monitor pulsed-field electrophoresis of large DNA fragments on-line might further improve the efficiency
of separations without a priori knowledge of the fragment sizes present, as is currently required for optimal pulsedfield electrophoresis separations.5 In addition, an electrophoresis run could be terminated as soon as the separation was complete, instead of a t the end of an arbitrary period. While ethidium bromide fluorescence is often used to view small DNA fragments on-line (with mobility reductions from the dye of approximately 17%), fragments over 100 kilobase pairs (kbp) long are slowed unacceptably with the addition of the intercalating dye.6 Alternatives to ethidium bromide fluorescence detection include UV shadowing7 and phase contrast imaging.8 UV shadowing using a CCD camera with a 16 bit A/D converter has yielded 5-ng detection limits for Hind111 fragments of phage A. Ultraviolet shadowing7~9avoids the mobility perturbations for on-line detection applications but does not eliminate the exposure to ultraviolet light. Phase contrast imaging has not been extensively applied to nucleic acid detection. We have previously shownl-3 that electric birefringence can be used to image nucleic acids in electrophoresis gels. No staining is required, and the technique works with visible light. It is necessary, however, that an electric field be applied to the gel. The usual electrophoresis voltages are adequate. Two phenomena generate image contrast.132 For large fragments, orientation of the nucleic acid fragment (Kerr effect) is the major contributor. The observed image resembles a conventional fluorescence image in morphology. For smaller fragments, the dominant effect is electrokinetic orientation of the agarosegel itself. In addition, there is always a small background from the Kerr effect of the agarose. Electrokinetic gel orientation generates images whose intensity follows the nucleic acid concentration gradient. Kerr effect images are independent of the polarity of the applied field. The two halves of the electrokinetic image change position when the electric field is inverted. Our earlier papers describe the basic phenomena of EBI. Although we have used EBI to visualize separated nucleic acid fragments for isolation and further experiments,'O we have not systematically investigated imaging aspects of the technique. In the present manuscript we describe techniques for maximizing image contrast and detectability. In addition, we briefly examine the effect of the wavelength of light, temperature, and orientation dynamics on image quality. (5) Noolandi, J. In Electrophoresis of Large D N A Molecules: Theory and Applications;Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1990. (6) Maniatus, T.; Fritach, E. F.; Sambrook, J. Molecular Cloning: A Laboratory Manual; Cold Spring . -Harbor Laboratory: ColdspringHarbor, New York, 1982. (7) Chan, King C.; Koutny, Lance B.; Yeung, Edward S. Anal. Chem. 1991,63, 746-750. (8)Pickett, S . C.; Jonston, R. F.; Miller, M. F.; Barker, D. L. Am. Lab. 1990, 22, 34-40. (9) Hassur, Steven M.;Whitlock,Howard, W., Jr. Anal. Biochem. 1974, 59, 162-164. (10) Demana, Tshenge, D.; Lanan, Maureen;Morris, Michael D. Anal. Chem. 1991,63, 2795-2797. ~~
(1) Lanan, Maureen;Shick, Reed, A,;Morris, Michael D. Biopolymers 1991,31, 1095-1104. (2) Lanan, Maureen; Morris, Michael D. Appl. Theoret.Electrophoresis 1992, 3, 27-32.. (3) Lanan, Maureen;Grossmann, Daniel W.; Morris, Michael D. Electrophoresis, submitted for publication. (4) Anand, Rakesh; Villasante, Alfredo; Tyler-Smith, Chris. Nucleic Acid Res. 1989,17, 3425-3433.
0003-2700/92/0364-1967$03.00/0 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1 , 1992
BACKGROUND It is possible to separate the Kerr effect and electrokinetic effect contributions to the birefringence images computationally. Depending upon the component displayed, the recovered images will have either a peaked shape (Kerr) or a bilobed shape (electrokinetic). The preferred technique will depend upon the relative contributions to the image. The sign of the electrokinetic birefringence depends on the direction of the electric field, while the sign of the Kerr effect contribution does not. Equation 1 describes the electrokinetic birefringence,' 6EK
= kEVC
(1)
where 6 is the optical retardance, k is a lumped constant, E is the electric field strength, and C is the DNA distribution function. While the electrokinetic response depends on the concentration gradient, the Kerr effect signal tracks concentration directly. Equation 2 describes the Kerr effect birefringence.
kE2C The overall electric birefringence is given by eq 3. The total optical retardance consists of Kerr and electrokinetic contributions arising from both gel and sample. 6 6KERRDNA 6KERRCEL + + 6EKGEL (3)
+
The different electric field polarity dependences of the Kerr and electrokinetic effect signals allow the two types of signals to be separated. To obtain the pure electrokinetic image, two consecutive images obtained with the electric fields reversed are subtracted (eq 4). For quantitative work each I E K = p -r (4) image, IX,is normalized (or flat-fielded) by the background light intensity before subtraction (eq 4). If the field-on times are the same for the two images and the images are properly normalized, the resulting difference image should contain only the electrokinetic contribution, PK. To obtain the pure Kerr effect image, F E R R , the same two images are added. From this sum, the image obtained under field-off conditions, IO, is subtracted. The procedure is described by eq 5. Any electrokinetic signals should be completely eliminated by this process, but the gel Kerr effect signal remains as an approximately constant background.
PERR = I+ + r - 2(P)
(5)
EXPERIMENTAL SECTION Buffers were prepared from stock solutions after filtering through 0.45-pm membrane filters to give 40 mM Tris-acetate, 1 mM EDTA, pH 8.2 ( 1 X TAE), or 45 mM Tris-Borate, 1mM EDTA, pH 8.45 (0.5X TBE), buffers. TBE stock was discarded after 1month to prevent precipitation. Stock ethidium bromide (Sigma) was dissolved in deionized water (6.8g/L) and stored at room temperature. Loading buffers tested consisted of various combinations of 2 X TAE, 0.5% SDS, 0.15% bromophenol blue, and 50% glycerol. These were added as 20% of the total sample volume. Hind111 fragments of X phage (BRL) were heated for 10min in BRL fragment buffer before being adjusted to l x TAE or 0.5X TBE, 6% sucrose. X DNA was the kind gift of Dr. Mitch Drumm from the Howard Hughes Research Institute at the University of Michigan. X DNA was diluted with electrophoresis buffer and made 6% sucrose before loading. All reagents were electrophoresis grade. The chemically modified agaroses SeaPlaque (hydroxyethylated agarose derived from G r a c i l a r i a ) and FastLane (low electroendoosmosis agarose derived from Gelidiurn) (FMC Bioproducts) were used. Following a standard protoco1,B agarose was dissolved in buffer using a stirring hot plate. After the agarose
was cooled to 65 "C, 20-40 mL was cast directly in the electrophoresis cell. Thicknesses ranged from 3 to 6 mm. Gels formed in 30 min at room temperature. Once solidified, the gel was covered with a few millimeters of buffer and stored at room temperature until use. The electrophoresis cell followed the design of Shafnefl except that an optically flat Pyrex glass-bottom plate was used in place of the conventional transparent plastic window. Buffer in polypropylenetubing was recirculatedwith a peristaltic pump through a chilled water bath. The temperature within the cell was measured with a type k thermocouple and recorded by computer after amplification with an AD595 thermocouple amplifier. Electric field strength was measured with platinum probes placed 1.2 cm apart and placed directly in the cell. Standard dc electrophoresis was performed with a BioRad Model 250/2.5 power supply with 4-8 V/cm applied to the gel for 1-2 h. For FIGE, a locally constructed relay system was used to invert the polarity of the applied voltage from the Model 250/2.5 power supply. FIGE was carried out for 5-10 h using a continually cyclinglinear ramp of forward and reverse voltage pulse durations. A constant 3:l forward to reverse voltage ratio was maintained for all pulse times. Reverse voltage pulses varied from 0.1 to 10 s. EBI was performed with the instrumentation previously However, for some experiments the incandescent lamp and 650-nm, 20-nm-band-pass, interference filter illumination system was replaced with a 70-W high-pressure sodium lamp and 589-nm,10-nm-band-pass,interference filter. In these experiments, a Polaroid 140 f 20-nm plastic film quarter-wave plate was employed. For dc electrophoresisa background image was acquired, before the start of the separation, with the electric field off. In FIGE, a pair of images acquired with opposite aligning voltages were recorded at intervals during the separation. To ensure that fragments were in the same position during each image of an FIGE cycle,integration was initiated at two-thirds of the duration of the longest positive voltage pulse and at the end of the matching reverse voltage pulse. No interruption of the FIGE separation protocol was necessary. In experiments where no net movement of fragments was desired, voltage pulses of opposite polarity but equal duration and depth were applied to the gel. Images were recorded halfway through each pulse. Imageswere compensatedfor light source intensity fluctuations by normalizing the entire image either by the average intensity of a gel-free region of the image or by dividing by the average image intensity. After compensation, images were processed according to eq 4 and/or 5 on a 386/20-MHz personal computer using programs written in Turbo Pascal (Borland). Further processing was done on a Macintosh Ilcxusingthe program Image 1.43." Preprocessed images were median-filtered to reduce spike noise associated with dust and pixel to pixel intensity variations. Birefringence signals are presented as the raw intensity values and are not reduced to optical retardance. For quantitative measurements,transverseaverageswere taken across the width of each electrophoresis band. The peak-peak height and peak areas of the averaged bands were used to develop working curves and define signal/noiseratios and detection limits. Peak areas were determined using standard protocols in Image.
RESULTS AND DISCUSSION Electric birefringence12 measures the change in ellipticity of transmitted light. The quarter wave plate is adjusted to 135' from the electric field direction. Nominally, the polarizer and analyzer are 90' to each other, with the polarizer at 45' to the electric field direction. It is necessary to rotate the analyzer slightly away from 90' to compensate for stray light from polarizer imperfections and strain birefringence in the apparatus. This rotation generates a background which must be subtracted from the measured signal. The background is determined by the bias angle of the analyzer. The (11) Rasband,Wayne.Image, Ver. 1.43;NIH ResearchServicesBranch Bethesda, MD, 1991. (12) Fredericq, E.; Houssier, C. Electric Dichroism and Electric Birefringence; Claredon Press: Oxford, U.K., 1973.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992 0.6
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Flguro 1. Effect of temperature regulatlon on EBI relative average Intensity. The gel was 1 % Seaplaque agarose In 1 X TAE. All data were scaled to 10 V/cm. Curve a was recorded without cooling at temperatures which Increased from 27 to 36 "C, (b) was at 26 "C, and (c) was at 15 "C.
Figure 2. Dose response curve for data from one gel evaluated as the flelbon minus fiekloff image. The DNA concentration was determined on the basis of the total concentrationof DNA loaded and by assumlng equimolar quantities of each HlndIII fragment of phage
bias angle is 6", which is appropriate for our stray light constant13 of 2 X 10-4. The average electric birefringence intensity of the blank gel is temperature dependent. Figure 1 shows the average birefringence intensity versus time for blank SeaF'laque agarose taken a t different temperatures and corrected to the same electric field strength. Curve a shows that without cooling, agarose birefringence increases by as much as 50%. Temperature continues to increase from 27 to 39 "C during this experiment while the blank gel intensity reaches an apparent maximum. With the gel maintained a t 26 "C, curve b shows a decrease in intensity of less than 10% from baseline. When the temperature is regulated at 15 "C and scaled to account for field strength differences between runs, curve c, the decrease is very similar to that found at 26 "C. With the temperature controlled the gel shows positive birefringence. The blank gel birefringence does not invert sign on reversing the electric field polarity in these experiments. We have tested the effect of buffer type, pH, temperature, and illumination wavelength on the signal from HindIII fragments of phage A. For quantitative comparisons,we have normalized the peak-peak height of the 23 kbp band EBI signal to the light source intensity and to the electric field strength. Variation of pH from 8.2 to 8.7 in TAE buffer has little effect, nor does changing the buffer from 1 X TAE to 0.5X TBE. We observe a 20% increase in birefringence when the temperature is reduced from 25 to 15 "C, consistent with the increase in refractive index. Substitution of a sodium vapor lamp (590 nm) for a filtered tungsten lamp (650 f 20 nm) gives an additional 9 % increase in birefringence which results from refractive index dispersion. Overall, we find close to a 30% improvement in signal by using a sodium lamp with gels at cold, regulated temperatures. However, without special reflectors commerciallyavailable tungsten flood lamps provide more intense illumination of the gel than do available sodium lamps. In addition, the CCD detector is somewhatmore sensitive a t 650 nm compared to a t 590 nm." To maintain a 2-5 exposure time with our apparatus, it is necessary to open the camera lens iris from f / l l to f / 4 on changing from tungsten to sodium lamp illumination. In general,the choice of illuminatingwavelength will be governed by the properties of other components of the optical train, including polarizers, lenses, and detectors. If the ultraviolet region is avoided, there appears to be no
intrinsically preferable wavelength range for EBI measurement. The loadingbuffer is not critical to the quality of the images. There is no observable effect on birefringence from adding SDS, glycerol, or sucrose to the loading buffer. However, bromophenol blue, a commonly used tracking dye, absorbs in the 590-670-nm region and obscures some regions of the EBI image if it is present. EBI response is uniform across a gel. We observe the same signal intensity across the gel loaded with equal amounts of DNA in each of the five wells, with no apparent systematic position effects. Similar results have been obtained in preparative experiments where one well has covered the entire width of the image. In early work,l EBI data were computed as the difference between images recorded with the electric field on and off (I = I+- lo). Figure 2 shows dose-response curves for electrokinetic images (I= I+-lo) of HindIII fragments in the size range 2-23 kbp and for loadings down to 10 ng. As expected for fragment lengths with signals caused primarily by electrokinetic effects,2 the response at a given field strength depends only upon the amount of nucleic acid present and not upon its size. The slope of the curve is directly proportionalto field strength,as predicted from theory.zThese data show that useful EBI response is obtainable at realistic loadings, typically about 20 ng/band minimum, and operating voltages, usually in the range 2-10 V/cm, without signal averaging or further processing. Figure 3 shows the effect on image contrast of two methods of background subtraction. In image a, the background is simply the image obtained with the electric field off. The background image should contain birefringencecontributions from gel irregularities and the much smaller irregularities in strain birefringence in the apparatus. Subtraction should remove these artifacts from the recovered image. This technique yields an image which contains contributions from the Kerr effect of agarose itself, the Kerr effect of the nucleic acid, and electrokinetic orientation of the agarose. Only the last two contribute to a nucleic acid image. Contrast is relatively poor, because the gel Kerr effect is not negligible and is not completely uniform throughout the image. In image b a pure electrokinetic image is obtained using the procedure of eq 4. The background is the image obtained by reversing the applied electric field. Strain birefringence in the apparatus is unaffected, but the technique gives higher contrast because it removes the large birefringence contribution of the agarose Kerr effect. Reversing the field direction leaves the sign and magnitude of the agarose and DNA Kerr effects unchanged. However, the signs of the two lobes of the electrokinetic image reverse. Subtraction, therefore, cancels
(13) Howier, C.;OKonski,C. T. InMolecuEarElectro-Optics: ElectroOptical Properties of Macromolecules and Colloids in Solution; Krause, S., Ed.; Plenum Press: New York, 1981;pp 309-339. (14)Bed, G.;Boucharlat, G.; Chabbeal, J. Opt. Eng. 1987,26, 902910.
A.
ANALYTICAL CHEMISTRY, VDL. 64, NO. 17, SEPTEMBER 1, 1992
1970
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b r a 3. Image processing effect on contrast. Images are the average of four images where (a)is processed as the field-an minus fbldaffimages and (b) is according to eq 4. Curves c end d are the transverseaverages of imagesa and b. respectively. Seaplaque agarose (1%) in 0.5X TBE wnh 200 ng total of HindIII was imagad at 5.9 Vlcm using 650-nm illumination.
"T
Fbure 5. Electric birefringence image of a 1% Seaplaque agarose gel. 1X TAE. The image is the average of four images recorded at 8.2 Vlcm and processed according to eq 4. The pattern in the 100-ng laneat the 2 kbp fragmentband was caused by dust. The 23 kbp band of the lane containing a total of 10 ng of DNA represents 4.8 ng of DNA.
concentration response is maintained for images processed by eq 4, as shown in Figure 4. Figure 5 shows the result of averagingfour successiveimages acquired in only 1min. There is sufficient contrast to allow detection (SIN 3) of 7.5 ng of the 23 kbp HindIII fragment based on peak to peak height. The results are comparable to those obtainable by UV shadowing.7 However, these data are obtained with a camera equipped with a 12-hit AID converter. Because EBI images are made by difference measurements, the detection limit depends partlyor entirely on AID converter resolution. Substitution of a 16-bit resolution camera should decrease detection limits by as much as a factor of 24. The noise in our gels is partly caused by gel inhomogeneities which produced local variations in blank gel signal. Unlike quantization noise, this noise source is not eliminated by increasing camera AID converter resolution. The detection limits we calculate are estimated using the noisiest adjacent section of blank gel, in order to give a conservativeestimate. A closer look at the image in Figure 5 indicates that a band containing 4.8 ng of 23 kbp DNA can actually be visualized. This observation suggeststhat the combination of appropriate digital filters and high AID converter resolution could substantially reduce the detection limits reported here. The background subtraction technique in eq 4 cannot completely compensate for bulk creep of the agarose gel. During long applications of electric field, bulk fluid flow distorts the gel. Because the creep direction depends on the sign of the electric field, creep is not compensated for. The effect is visually clearer in the transverse average plots of Figure 3c,d of the data in the images in Figure 3a,b. In both plots creep appears as a slowly undulating background. Because the creep background contains only very low spatial frequencies, visual examination of the nucleic acid images is not adversely affected. Electrokinetic orientation gives bilobed nucleicacidimages in place of the more familiar peak-shaped images resulting from fluorescenceor the nucleic acid Kerr effect. If visualization of nucleic acid is the aim of the experiment, then the bilohedimagesareperfectlysatisfactory.The bandis defined by the return to the background intensity, just as in peakshaped images. There may even be some advantage to the use of bilobed images in mapping applications because the zero-crossing permits easier definition of the band center. The choice of background correction technique to isolate either a Kerr effect or electrokinetic image depends upon their relative contrihutions. The Kerr effect signal can be isolated by adding the images obtained with the field on in
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both the DNA and agarose Kerr effect signals but leaves an electrokineticimagewhose contrast isabout 2.6X greaterthan that in image a. Twenty consecutive images were acquired during the experimental sequence which includes the four runs used to generate Figure 3. The signal to noise ratio based on peaktopeak height attained from image to image fluctuates. For one image, however, the SIN ratio obtained by simply subtracting the field-off image is 11zk 4. (Noise is defined as the standard deviation of a blank region of the transverse average plot.) When the data are processed according to eq 4, the S/N is 29 f 8. Extrapolating to SIN = 3, the detection limit for the 23 kbp DNA fragment band is estimated at 10 ng. For dc electrophoresis on-line detection of small samples suffers because the field cannot he reversed. If the field can be reversed, as in FIGE or in a modified de protocol, then the sensitivity of on-line data acquisition can be enhanced. In any case, signalaveraginganddetection withreversingelectric fields is possible after the dc separation is complete. Integrated peak area can also be used for quantitation. Peak areas for EBI signals from HindIII fragments loaded per lane with a total of from 10 to 400 ng of DNA were determined and plotted in Figure 4. The signal to noise ratio is similar to that observed for peak-peak height measurements. For two different gels run on different days, the detection limit (SIN = 3) is 13 i 2 ng of DNA per 62-pixelwide lane. This represents an improvement by a factor of -2 compared to subtraction of the field-off image. Linear
ANALYTICAL CHEMISTRY, VOL. 64. NO. 17. SEPTEMBER 1, 1992
1971
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mura 0. Transverse average plots of Images compare EBI signal processlng techniques for Hinull1 fragments of phage A to the fluorescence Intensky proflle. SeaPlaque agarose (1%) at 16.5 V I cm. 26 O C was used. A total of 170 ng of DNA was loaded. Curve a is the Kerr effect Image processed according to eq 5. Curve b is me fluorescenceintensky profile. Curve c is the electrokinetic image processed according to eq 4. Curved is the numerical integration of curve c. A fltmorder pobnomlal was in to the raw integrated fileand subtracted to flatten the baseline kbp'
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Fbure 8. Effect of image processing on unresolved Mbp DNA fragments. Image a shows the Kerr effectimage and image b shows theelectrddneticimage. See Figure 7 caption for experlmental detalls.
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Figure 7. Effect of data processing on 48 end 96 kbp DNA fragments. Images were acquired during an F I E separation at 9.6 Vlcm in B-mmthlck 1% FastLane agarose, 0.5X TEE, 9 "C. containing 200 ngtotalof ADNA. Theelectrokineticlmage(b)and transverseaverage plot (a)are shown together wnh the Kerr effectImage (c)and average plot (d). the forward and reverse directions. For Hind111 fragments in SeaPlaque, the electrokinetic contribution is larger. The transverse average plots in Figure 6 show this behavior clearly. In these plots Kerr effect and electrokinetic images are compared to an ethidium fluorescence image. Of course, it is also possible to numerically integrate the electrokinetic image to recover the familiar peak-shaped response of fluorescence, absorbance, or Kerr effect imaging. As shown in Figure 6d, the integration procedure also improves the signalinoise ratio of the plotted data. With heavier fragmentsthe Kerr effect of DNA contributes more strongly to the observed image. This can be seen in Figure 7, which shows the Kerr effect (c) and electrokinetic (b)imagesofthe48and96khpfragmentsof250ngofADNA inFastLane agarose.0.5X TBE. Thetransverse average plots (a and d) demonstrate that the signal contains significant amount of both electrokinetic and Kerr effect components. Fractional orientation of DNA fragments in an electric field increases with fragment size. Data processing by eq 5 does not compensate for gel Kerr effect signals. Local variations in the gel Kerr effect signal may account for the undulating baseline seen in Figure ICnear the small-fragment region. Electrokinetic signals also decrease as hands broaden, decreasing the spatial concentration gradient. For unresolved Mbp fragments, the Kerr effect signal dominates, as can be seen in Figure 8,
Dynamics of electrokinetic and Kerr effect signals for 48 Transverse average plots acquired using voltage pulses increasing by 10saredisplayed. SeeFigure 7 captionforexperlmental details. Flgurs B. kbp DNA.
Figure IO. Peak to peak height of tha elemokinetlc signal versus voltage pulse duration fw the system In Figure 9.
EBI response depends on the relative dynamics of electrokinetic and Kerr effect responses for a given fragment length. Iftheorientingelectricalpulaeis tooshort, the system may not have sufficient time to align, causing weak EBI signals. Data evaluation hy eq 4 and 5 permits separation of the intrinsic Kerr response and the induced electrokinetic response. Figure 9 represents transverse average plots of imagesof A DNA (48khp) acquired using voltagepulses which increase in length from 10 to 30 8. The peak-to-peak height for the electrokinetic signal increases for 40 s before reaching steady-stateintensity (Figure 10). Incontrast,theKerreffect signal maximizes within 10 s of the applied pulse.
1972
ANALYTICAL CHEMISlRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
CONCLUSIONS EBI imaging is a simple and useful technique for visualizing nucleic acids in agarose electrophoresis gels. The contributions to EBI have been modeled successfully, and the theory provides the baais for satisfactory background corrections in analytical EBI applications. From observed EBI signals at all fragment lengths, purely electrokinetic signals and purely intrinsic birefringence signals can be extracted. Either signal can be used but the preferred technique depends on which contribution is stronger. Because EBI is inherently a difference technique, the ability to determine differences is directly related to the dynamic range of the camera and associated AID converter. With a CCD fitted with a 12-bit converter, differences of one part in 4000 can be observed. Even so, the detection limits compare favorably with those obtained by
UV shadowing usina a camera fitted with a 16-bit A/D converter and 81.e only a factor of 5-6X greater than detection limits for ethidium staining. Alternatively, for preparative applications where loadings above 100 ng are employed, the greatest possible detectivity is not necessary and it may be adequate to employ a less expensive camera with an 8-10-bit AID converter.
ACKNOWLEDGMENT This work was supported by NIH Grant GM-37006.
RECEIVED for review January 16, 1992. Accepted May 21, 1992.
