Anal. Chem. 1999, 71, 5003-5008
Miniaturized Ultrathin Slab Gel Electrophoresis with Thermal Lens Microscope Detection and Its Application to Fast Genetic Diagnosis Jinjian Zheng,† Tamao Odake,‡ Takehiko Kitamori,†,‡ and Tsuguo Sawada*,†
Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan, and Kanagawa Academy of Science and Technology, 3-2-1 Sakado, Takatsu, Kanagawa 213-0012, Japan
A miniaturized ultrathin slab gel electrophoresis (MUSGE) apparatus was developed, and fast separation of DNA fragments was obtained using it. To obtain sufficient separation efficiency in a limited space, a discontinuous buffer system was used. In general, it is difficult to cast a discontinuous ultrathin slab gel of adequate quality. However, the miniaturized resolving gel could be cast by taking advantage of the “capillary phenomenon” of the ultrathin channel. A gradient plate was used to control the height of the resolving gel and to form a clear interface between the concentrating gel and the resolving gel. This method was used to cast multiple gels simultaneously and reproducibly. The gradient plate also facilitated sample introduction, which was carried out by using a micropipet. A 25-mm-long and 80-µm thick-resolving gel was used to separate the 100-base pair ladder DNA within 10 min. Bandwidth was reduced to 100-200 µm, thus improving the number of theoretical plates to 22 000, which was comparable to that in conventional slab gel electrophoresis even though the migration distance was reduced to 1/10. Satisfactory lane-to-lane reproducibility (RSD < 1.0%, n ) 6) and gel-to-gel reproducibility (RSD < 2.7%, n ) 4) were obtained. Finally, the MUSGE apparatus was successfully applied to get a rapid genetic diagnosis. The Human Genome Project has stimulated the development of high-throughput DNA sequencing technologies such as ultrathin slab gel electrophoresis (USGE)1-3 and capillary array electrophoresis (CAE).4-6 At present, 500-600-base pair DNA fragments can be sequenced within 2 h. As a benefit from these * To whom all correspondence should be addressed: (fax) +81-3-5841-6037; (e-mail)
[email protected]. † The University of Tokyo. ‡ Kanagawa Academy of Science and Technology. (1) Chen, D.; Peterson, M. D.; Brumley, R. L.; Giddings, M. C.; Buxton, E. C.; Westphall, M.; Smith, L. M. Anal. Chem. 1995, 67, 3405-3411. (2) Carninci, P.; Volpatti, F.; Schneider, C. Electrophoresis 1995, 16, 18361845. (3) Smith, L. M.; Brumley, R. L.; Buxton, E. C.; Giddings, M.; Marchbanks, M.; Tong, X. Methods Enzymol. 1996, 271, 219-237. (4) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 21492154. (5) Ueno, K.; Yeung, E. S. Anal. Chem. 1994, 66, 1424-1431. (6) Dovichi, N. J. Electrophoresis 1997, 18, 2393-2399. 10.1021/ac990408i CCC: $18.00 Published on Web 09/27/1999
© 1999 American Chemical Society
technologies, the total 3 billion base human genome is expected to be completely sequenced by 2003.7 Already attention is being focused on the study of genetic variation among individuals. The most common type of human genetic variation is single-nucleotide polymorphisms (SNPs),8,9 which occur on average once per 750 bases of human genome. Therefore, detection of SNPs should provide a powerful tool for human genetic studies. SNPs have been detected using a DNA hybridization chip.10,11 However, problems in mismatch of the hybridization reaction have to be overcome.12 On the other hand, direct sequencing using USGE and CAE should provide more accurate information. For detection of SNPs, the required read length is only 100-150 bases. Therefore, it is not necessary to employ current USGE and CAE instruments, which were mainly developed for long read-length DNA sequencing and are relatively expensive. Several groups have reported that separations with enhanced speed and reduced cost could be obtained by using microfabricated CAE chips.13,14 We proposed that fast and inexpensive separations could also be obtained by miniaturized ultrathin slab gel electrophoresis (MUSGE). Miniaturization of USGE and CAE should provide the following advantages. First, high speed can be obtained due to the advantages of combining short separation distances and a high electric field. Second, the quantities of reagents can be reduced, resulting in a lowered cost. Third, even a low voltage will result in a high field, thus reducing the cost of the electrical power supply, as well as the hazard of electrical shock. Furthermore, in MUSGE, the apparatus is relatively simple, and technologies developed so far for USGE can be used with little or no modification. However, several problems exist in miniaturizing USGE, including how to improve the system efficiency in a limited space (7) (8) (9) (10) (11)
(12) (13) (14)
Marshall, E. Science 1998, 281, 1774-1775. Risch, N.; Merikangas, K. Science 1996, 273, 1516-1517. Gu, W. K.; Aguirre, G. D.; Ray, K. Biotechniques 1998, 24, 836-837. Vo-Dinh, T.; Alarie, J. P.; Isola, N.; Landis, D.; Wintenberg, A. L.; Ericson, M. N. Anal. Chem. 1999, 71, 358-363. Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. J. Nucleic Acids Res. 1997, 25, 49074914. Fotin, A. V.; Drobyshev, A. L.; Proudnikov, D. Y.; Perov, A. N.; Mirzabekov, A. D. Nucleic Acids Res. 1998, 26, 1515-1521. Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181-2186. Schmalzing, D.; Adourian, A.; Koutny, L.; Ziaugra, L.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 1998, 70, 2303-2310.
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and how to overcome the difficulties in carrying out sample introduction and gel construction. The separation efficiency can be improved by using a high electric field15 and a discontinuous buffer system.16,17 Use of a high field will reduce band broadening from molecular diffusion, thus improving the separation efficiency. In MUSGE, it is easy to apply a high electric field because (1) the undesired Joule heat can be dissipated efficiently due to the use of an ultrathin slab gel and (2) a high field can be obtained by using a low-voltage electrical supply. The discontinuous buffer system is able to concentrate the injected samples on-line, thus reducing band broadening and improving the separation efficiency as well. It is often used for electrophoretic separation of protein compounds18,19 and can also be used for the separation of DNA fragments as we have described in a previous study.16 Therefore, the separation efficiency should be further improved by combining MUSGE and a discontinuous buffer system. However, constructing a discontinuous ultrathin slab gel has proved to be much more difficult than constructing a conventional continuous slab gel because it is difficult to obtain a clear interface between the concentrating gel and the resolving gel at a desired place. Another problem is that sample introduction in ultrathin slab gel electrophoresis is difficult, and this is the same in MUSGE. To overcome this problem, several groups have reported various methods. Stein et al.20 used a sharp comb fashioned from a single-edged razor blade by spark erosion using an electric discharge machine. Liu and Sweedler21 and Hietpas et al.22 developed a sample introduction system using a capillary. However, implementing these methods is rather complicated, and the applicable sample volume is limited. A sample introduction method, which is simple to implement, and allows a flexible sample volume, is desirable. In this work, we developed a miniaturized vertical ultrathin slab gel electrophoresis apparatus, which was ∼1/10 the size of a conventional slab gel. A discontinuous buffer system was used to concentrate the injected sample, thus reducing band broadening and improving the separation efficiency. A unique gel-casting device taking advantage of the “capillary phenomenon” of the miniaturized ultrathin channel was developed, and the discontinuous ultrathin slab gel was easily constructed. This gel structure also facilitated sample introduction. To detect the separated bands, we used the thermal lens microscope (TLM) detector,16,23-25 which has high sensitivity and high spatial resolution, in combination with a simplified silver staining method. The performance of this miniaturized system was evaluated by separating 100-base pair (15) Mastrangelo, C. H.; Burns, M. A.; Burke, D. T. Proc. IEEE 1998, 86, 17691787. (16) Zheng, J.; Odake, T.; Kitamori, T.; Sawada, T. Anal. Sci. 1999, 15, 223227. (17) Doktycz, M. J. Anal. Biochem. 1993, 213, 400-406. (18) Ornstein, L. L. Ann. N. Y. Acad. Sci. 1964, 121, 321-349. (19) Chrambach, A.; Jovin, T. M. Electrophoresis 1983, 4, 190-204. (20) Stein, A.; Hill, S. A.; Cheng, Z. Q.; Bina, M. Nucleic Acids Res. 1998, 26, 452-455. (21) Liu, Y. M.; Sweedler, J. V. Anal. Chem. 1996, 68, 2471-2476. (22) Hietpas, P. B.; Bullard, K. M.; Gutman, D. A.; Ewing, A. G. Anal. Chem. 1997, 69, 2292-2298. (23) Harada, M.; Iwamoto, K.; Kitamori T.; Sawada, T. Anal. Chem. 1993, 65, 2938-2940. (24) Harada, M.; Shibata, M.; Kitamori T.; Sawada, T. Anal. Chim. Acta 1995, 299, 343-347. (25) Mawatari, K.; Kitamori, T.; Sawada, T. Anal. Chem. 1998, 70, 5037-5041.
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Figure 1. Construction of the discontinuous miniaturized ultrathin slab gel.
DNA ladders and λ DNA-HindIII/ΦX-174 DNA-HincII digest. Finally, we applied this system to a genetic diagnosis of coronary heart disease. EXPERIMENTAL SECTION Gel Construction. The channel for casting the miniaturized vertical ultrathin slab gel was constructed by using two quartz plates and two Teflon spacers, 45 mm long × 6 mm wide × 80 µm, thick as shown in Figure 1a. This channel consisted of two parts. The lower one, which was 25 mm long × 28 mm wide × 80 µm thick, was used to cast the resolving gel. The upper one, which was 10 mm long × 28 mm wide and gradated in thickness from 80 to 680 µm, was used to cast the concentrating gel. This could be easily achieved by partially beveling the notched quartz plates. A ceramic comb, which was fashioned from a 1-mm-thick ceramic plate to fit exactly into the gradient channel, was used to form the wells. Both the width and the spacing of the comb teeth were 1 mm and the tooth tips were 350 µm thick. The most important aspect in preparing a miniaturized ultrathin slab gel is elimination of particles and lints, grease residues, and other contaminants on the plates or spacers since they are potential sites for the formation of air bubbles which would interfere with the electrophoresis. In this work, the plates were first cleaned with alkali detergent, rinsed with Milli-Q water, and then immersed in H2SO4/H2O2 solution (3:1) for 10 min at room temperature. The plates were rinsed with flow Milli-Q water for 10 min and then with ethanol and left to dry in a clean case. The Teflon spacers were wiped with acetone and a lint-free wiper. To carry out silver staining after electrophoresis, the slab gel has to be bound to the plane plate and to be separated easily from the notched plate. Prior to gel casting, the notched plate was wiped with repel silane (20 g/L dimethyldichlorosilane solution in 1,1,1-
Figure 2. Schematic illustration of the thermal lens microscope.
trichloroethane), left to dry for 5 min, and then wiped with a lintfree wiper. The plate was then rinsed with water and 95% ethanol and dried. This treatment made it easy to remove the notched plate from the slab gel. On the other hand, the plane plate was immersed for 1 h in a 0.8% (v/v) bind silane ([γ-(methacryloxy)propyl]trimethoxysilane) solution, which had been dispersed into a pH 3.5 acetic acid solution and thoroughly stirred until clear. The plate was then rinsed with Milli-Q water and 95% ethanol and dried. This treatment bound the slab gel to the plate and ensured it did not separate during silver staining. The gel casting cassette (Figure 1a) was placed vertically in a shallow plastic dish as shown in Figure 1b. After degassing and adding polymerization initiators, the resolving gel solution was poured into the plastic dish. Because of the “capillary phenomenon”, the resolving gel solution rose into the ultrathin channel and stopped at a place just above the interface of the ultrathin channel and the gradient channel. After polymerization of the resolving gel solution, the concentrating gel solution containing initiators was poured into the gradient channel. A ceramic comb that had been fabricated to fit exactly into the gradient channel was inserted. When the concentrating gel had completely polymerized, the comb was withdrawn carefully, leaving sampling wells in the gel. Gel Components. The discontinuous buffer system used in this experiment contained Cl- ion as the leading ion and glycine ion as the terminating ion. Both the resolving gel buffer and the concentrating gel buffer contain the same leading ion but with different pH. The buffer solutions were made according to the following process: (1) The resolving gel buffer, 0.15 M pH 8.8 Tris-HCl, is made by dissolving 18.2 g of Tris in Milli-Q water, adjusting the pH to 8.8 with 4 M HCl, and then making up to 1000 mL with Milli-Q water. This buffer solution is also used as anode buffer. (2) The concentrating gel buffer, 0.1 M pH 6.8 TrisHCl, is made by dissolving 12.1 g of Tris in Milli-Q water, adjusting the pH to 6.8 with 4 M HCl, and then making up to 1000 mL with Milli-Q water. (3) The cathode buffer contains the terminating ion, glycine ion. It is made by dissolving 3.0 g of Tris and 14.4 g
of glycine with Milli-Q water and making up to 1000 mL with Milli-Q water. A 7.5% T, 3% C acrylamide/N,N′-methylenebisacrylamide solution containing the resolving gel buffer was used to cast the resolving gel. A 3% T, 3% C acrylamide/N,N′-methylenebisacrylamide solution containing the concentrating gel buffer was used to cast the concentrating gel. Both gel solutions were degassed under vacuum for 5 min at room temperature. Polymerization of the gel solutions was initiated by adding 8 µL of 10% ammonium peroxydisulfate solution (prepared daily) and 7 µL of 10% TEMED solution for 1-mL gel solutions. The anode buffer reservoir was filled with 0.15 M pH 8.8 Tris-HCl solution and the cathode buffer reservoir with 0.025 M pH 8.3 Tris-glycine solution. For conventional continuous slab gel electrophoresis, a 7.5% T, 3% C polyacrylamide gel with 0.1 M pH 8.5 Tris-borate buffer containing 1 mM EDTA (TBE) was used. This buffer solution is also used in anode and cathode reservoirs. TLM Instrumentation. A schematic illustration of the laboratory-constructed TLM instrument is shown in Figure 2. In this work, the emission line of an air-cooled argon ion laser (488 nm, 4 mW) was used as the excitation beam after mechanical chopping. The modulated beam was transmitted through a dichroic mirror, adjusted coaxially with the probe beam (633 nm, 1 mW) which came from a He-Ne laser, and was reflected off the same dichroic mirror. Both beams were introduced into an optical microscope and focused onto the surface of a gel using a 40× NA 0.65 objective lens. After silver staining, the slab gel was fixed onto a three-dimensional scanning stage which could be moved with a precision of 1 µm. TLM determination of the separated DNA fragments was achieved by driving the stage using a personal computer. Chemicals. The 100-base pair ladder DNA (1 ng/nL), λ DNAHindIII/ΦX-174 DNA-HincII digest (0.5 ng/nL), repel silane, bind silane, and silver staining kit were purchased from Pharmacia Biotech. Before injection, the DNA samples were diluted to proper concentration with loading buffer (0.08 M Tris-HCl, pH 6.8, and 0.01% Bromophenol Blue in 10% glycerol solution). Other reagents Analytical Chemistry, Vol. 71, No. 21, November 1, 1999
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Figure 3. The 100-base pair ladder DNA fragments separated in the discontinuous miniaturized ultrathin slab gel. Thickness of resolving gel, 80 µm. Applied voltage, 200 V (80 V/cm). Separation time, 10 min. Samples: from lane 1 to lane 6, 0.25 ng/nL for 9, 9, 18, 18, 37, and 37 nL, respectively. Components of gels were as described in the Experimental Section. The separation pattern was silver stained.
were of analytical or electrophoresis grades and were purchased from Wako (Osaka, Japan). Injection of more than 100-nL samples was performed by using a micropipet, and injection of less than 100-nL samples was performed by using a nanoinjector (Drummond). Safety concerns: Care should be taken when handling acrylamide monomers as they are toxic. RESULTS AND DISCUSSION Miniaturized Discontinuous Ultrathin Slab Gel. In this work, we constructed the miniaturized discontinuous ultrathin slab gels using the method described above. The resolving gel solution easily filled in the whole miniaturized ultrathin channel. Because occurrence of the capillary phenomenon decreased with the increased thickness of the upper channel in Figure 1a, the resolving gel solution stopped at the interface of the two parts and formed a clear surface. This gel-casting method makes it easy to cast multiple gels simultaneously, thus reducing the labor in casting gels and improving the gel-to-gel reproducibility. Using the ceramic comb described above, we obtained sampling wells whose entrances were larger than 1000 µm × 350 µm, allowing sample introduction to be carried out by a micropipet the same as for a conventional slab gel. This sample introduction method provides two advantages: (1) quantitative sample introduction is possible; (2) sample volume is flexible, ranging from several nanoliters to 1 µL. These advantages are very important for quantitative determination of samples even in a small volume. Evaluation of the MUSGE Separation System. Performance of this miniaturized system was evaluated through the separation of 100-base pair ladder DNA. An electric field of 80 V/cm was used, and six samples ranging from 9 to 37 nL were separated in 10 min. The results are shown in Figure 3. The 100-base pair 5006 Analytical Chemistry, Vol. 71, No. 21, November 1, 1999
Figure 4. TLM detection results of DNA fragments separated by MUSGE. Excitation beam: air-cooled Ar ion laser, 488 nm, 4 mW. Probe beam: He-Ne laser, 633 nm, 1 mW. Frequency of light chopper, 1076 Hz. Interval of 3D scanning stage, 10 µm/step. Time constant of lock-in amplifier, 200 ms.
ladder DNA samples were separated with an effective migration distance no longer than 14 mm. The separation speed was comparable to that in capillary electrophoresis and was 10 times faster than that in conventional slab gel electrophoresis. It should be noted that this result was obtained by using a voltage of only 200 V, contrasting with ∼2000 V in conventional gel electrophoresis and more than 10 000 V in capillary electrophoresis. The separation pattern in Figure 3 was then determined by TLM. The results are shown in Figure 4. DNA fragments of 100, 200, ..., 1500 bp were resolved to the baseline. DNA fragments longer than 1500 bp were not completely separated. We attributed this to the biased reptation of longer fragments, a general phenomenon in slab gel electrophoresis when a high electric field is used.26 The onset of this phenomenon shifted to 900-bp DNA fragments when an electric field of 120 V/cm was used. The bandwidth of separated fragments was determined to be 100200 µm, ∼1/10 of that in conventional slab gel electrophoresis. The number of theoretical plates of this miniaturized system was calculated as 22 000-24 000, comparable to that obtained in a conventional slab gel electrophoresis device. Lane-to-lane reproducibility was also demonstrated in this experiment. For example, the migration distances were very consistent from lane to lane, ranging from 13.7 to 14.1 mm for the 100-bp fragments (RSD < 1%, n ) 6). This was attributed to the unique gel construction method which takes advantage of the capillary phenomenon. A clear interface between the concentrating (26) Duke, T.; Monnelly, G.; Austin, R. H.; Cox E. C. Electrophoresis 1997, 18, 17-22.
gel and the resolving gel was formed, thus improving the laneto-lane reproducibility. Since multiple gels were cast simultaneously, by reducing the difference of timing in introducing gel solutions, the gel-to-gel reproducibility was improved too (RSD < 2.7%, n ) 4 for fragments from 200 bp to 1500 bp). Both the resolution and the reproducibility of this separation system were comparable to those in conventional slab gel electrophoresis, indicating that there are no obstacles to applying this method to treat samples that have so far been treated by conventional slab gel electrophoresis. The concentration limit of the injected sample was lowered to 10 pM (S/N ) 2). This is attributed to three aspects: (1) the concentrating effect of a discontinuous buffer system; (2) large sample volume with this gel structure; and (3) high sensitivity of TLM. Application of the MUSGE Separation System to Genetic Diagnosis. We applied the MUSGE to a genetic diagnosis of coronary heart disease. As shown above, DNA fragments ranging from 100 to 1500 bp were resolved to the baseline by MUSGE. Therefore, it should be useful for the determination of the variable number of tandem repeat (VNTR) alleles of human apolipoprotein B gene (apoB),27,28 which generally contain 25-60 repeat units of a 16-bp oligonucleotide and span from 500 to 1100 bp. Genetic linkage study has revealed that VNTR alleles containing more than 38 repeat units (707 bp) have a significant association with coronary heart disease.27 To determine the VNTR alleles, a resolution of 16 bp for DNA fragments ranging from 500 to 1100 bp is required, especially for fragments around 707 bp (38 repeat units). To study the suitability of this MUSGE system for analysis of VNTR alleles, first we applied it to the analysis of λ DNA-HindIII/ ΦX-174 DNA-HincII digest, a DNA ladder containing fragments ranging from 79 to 1057 bp, covering the size range of apoB VNTR alleles. The separation patterns are shown in Figure 5. The samples and electrophoresis conditions were the same, except that in Figure 5a a discontinuous buffer system was used, and in Figure 5b a continuous buffer system was used. For fragments ranging from 395 to 1057 bp, better resolution was obtained when the discontinuous buffer system was used. For example, the 612bp band and 770-bp band could not be completely separated using the continuous buffer system. However, they were completely separated using the discontinuous buffer system. Another advantage of the discontinuous buffer system was that it extended the limit of detection to a lower concentration due to its concentrating effect. For the low-concentration sample of lane 1, the faint 564bp band was not observed in Figure 5b, but it was easily observed in Figure 5a. Therefore, we concluded that MUSGE with the discontinuous buffer system, as described in the Experimental Section, was more advantageous for the analysis of apoB VNTR alleles. Fragments smaller than 345 bp were not separated in Figure 5a as well as in Figure 5b, but this did not affect the analysis of apoB VNTR alleles. If necessary, we can increase the concentration of the resolving gel or use a terminating ion with higher mobility instead of glycine to selectively separate these fragments. It should be noted that a 188-µm-thick resolving gel was used to (27) Friedl, W.; Ludwig, E. H.; Paulwever, B.; Sandhofer, F.; McCarthy, B. J. J. Lipid Res. 1990, 31, 659-665. (28) Baba, Y.; Tomisaki, R.; Sumita, C.; Morimoto, I.; Sugita, S.; Tsuhako, M.; Miki, T.; Ogihara, T. Electrophoresis 1995, 16, 1437-1440.
Figure 5. Separation of λ DNA-HindIII/ΦX-174 DNA-HincII digest. Thickness of resolving gel, 188 µm. Applied voltage, 150 V (60 V/cm). Separation time, 7.5 min. Samples: lane 1, 0.025 ng/nL for 1100 nL; lane 2, 0.1 ng/nL for 276 nL; lane 3, 0.25 ng/nL for 74nL; lane 4, 0.25 ng/nL for 110 nL. Components of the buffer systems as described in the Experimental Section.
Figure 6. Electropherograms of PCR-amplified apoB VNTR alleles. The gel components were the same as in Figure 5. The parameters of TLM detection were the same as in Figure 4. Sample volume, 200 nL. Electrophoresis, 80 V/cm for 8 min.
study this problem. When a 80-µm-thick resolving gel is used, the advantage of using a discontinuous buffer system becomes more clear. Almost no band can be observed with a continuous buffer system. The electropherograms of two apoB VNTR alleles samples are shown in Figure 6. The electrophoresis separation was finished in 8 min, half the time needed in capillary electrophoresis.28,29 One sample was determined to contain two VNTR alleles, 35 (659 bp) and 31 (595 bp) repeat units, indicating that this individual did not have a risk factor for coronary heart disease. The other sample was determined to contain 53 (947 bp) and 35 (659 bp) repeat unit alleles, indicating that this individual did have an increased risk factor. The results obtained here were consistent with those obtained by a standard method,29 showing that this method provides a reliable and efficient tool for genetic diagnosis of coronary heart disease. CONCLUSIONS In this work, we developed a miniaturized ultrathin slab gel electrophoresis apparatus that was ∼1/10 the size of a conventional slab gel electrophoresis device. Problems associated with resolu(29) Boerwinkle, E.; Xiong, W.; Fourest, E.; Chan, L. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 212-216.
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tion, sample introduction, and gel construction were studied. Compared with conventional slab gel electrophoresis, 10-fold faster separation was achieved by using a voltage of only 200 V. This system should be very useful in treating small-volume and lowconcentration samples because it allows a large range of volumes from several nanoliters up to 1 µL to be quantitatively injected. Twelve samples could be processed in parallel using this apparatus. It is easy to use wider plates to allow more samples to be processed in parallel, increasing the throughput of the miniaturized system. We demonstrated that TLM could provide high-sensitivity and high-spatial resolution for MUSGE. Currently, most laboratorymade TLM instruments are thought to be large and complex.
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However, this instrument can be compacted using the advanced small lasers, and the total size is only a bit larger than a conventional optical microscope. Such a TLM will become more compatible to the simple and small MUSGE system. ACKNOWLEDGMENT We are grateful to Dr. Yoshinobu Baba of Tokushima University who provided the PCR-amplified apoB VNTR allele samples.
Received for review April 20, 1999. Accepted August 15, 1999. AC990408I
