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1990, Yokohama, p. 412. 12 S. Funahashi, Y. Yamaguchi and M. Tanaka, Bull. Chem. Sot. Jpn., 57 (1984) 204. 13 S. Funahashi, K. Saito and M. Tanaka, Bull. Chem. Sot. Jpn., 54 (1981) 2695. 14 M. Tabata and M. Babasaki, Proceedings of the2nd Eurasia Conference on Chemistry in Seoul, April 16-20,1990, p. 336. 1.5 S. Funahashi, Y. Yamaguchi and M. Tanaka, Znorg. Chem., 23 (1984) 2249. 16 M. Tabata and M. Tanaka, J. Chem. Sot. Chem. Commun., (1985) 42. 17 M. Tabata and K. Oshita, Mikrochim. Acta, (1985) 397. 18 M. Tabata and N. Kajihara, Anal. Sci., 5 (1989) 719. 19 M. Tabata and K. Suenaga, Bull. Chem. Sot. Jpn., 64 (1991) 469. 20 M. Tanaka, Anal. Chim. Acta, 29 (1963) 193. 21 G. Nakagawa and M. Tanaka, Talanta, 19 (1972) 559. 22 S. Funahashi, Y. Ito, H. Kakito, M. Inamo, Y. Hamada and M. Tanaka, Mikrochim. Acta, (1986) 33.

23 Kinetic Aspects in Analytical Chemistry, International Chemical Congress of Pacific Basic Societies. Honolulu, Hawaii, Dec. 17-22, 1989, Abstract Nos. ANYL 0175-0181, 0209-0214,0245-0252,0272-0276. Dr. Masaaki Tabata is an associate professor at the Department of Chemistry, Saga University, Japan. He has worked with Professor Motoharu Tanaka since he was a master-course student at Nagoya University. His research interest is the reaction mechanism of the metalloporphyrin formation and the application of the results to the analysis of trace elements. Dr. Motoharu Tanaka is an emeritus professor of Nagoya University since 1989 and is currently a member of the Science Council of Japan. His research interests are equilibrium and structural studies on metal amino carboxylates; extraction of metal carboxylates; kinetic and catalytic method of analysis based on the mechanistic studies on reactions involving metal complexes. He has authored or co-authored more than 200 research papers and several monographs in the field of analytical and solution chemistry.

Detecting DNA bands in electrophoresis gels with chemiluminescence Margaret E. Casperson, and Edward M. Davis

Robert W. Coughlin

Wallingford, CT, U.S.A. A simple process is described for visualizing polynucleotides in agarose electrophoresis gels using a chemiluminescent label. Streptavidin conjugated to horseradish peroxidase was used to affinity-label biotinylated DNA. A light-emitting signal from affinity-labeled DNA was generated and recorded on photographic film after soaking the electrophoresis gel in a solution of peroxide and luminol.

Introduction Electrophoresis of DNA in gels of agarose followed by ethidium bromide staining of the DNA bands is a basic technique used in molecular genetics. For many years few improvements have been made on this simple and practical technique. This situation, however, has changed. Increased speed, convenience, safety, and sensitivity are now required to keep pace with high productivity demands being placed on those employing molecular biology techniques. The key impediment to these needed improvements is the use of the fluorescent dye, ethidiurn bromide, which is used to visualize DNA in agarose gels. Visualization by this method is relatively insensitive in that each band of DNA in the electrophoresis gel must contain 1 to 10 ng of DNA for de0165-9936/91/$03.00.

tection. Therefore, relatively large quantities of DNA must be loaded onto the electrophoresis gel. The use of special equipment for visualizing and recording DNA bands in electrophoresis gels is another disadvantage associated with ethidium bromide staining. An ultraviolet (UV) light source and a photographic system are both required. UV light, which excites the fluorophore ethidium bromide for visualizing stained DNA, is potentially dangerous to the researcher since prolonged exposure can cause burns. In addition, prolonged exposure of DNA to the UV light can damage DNA, photochemically cleaving it into smaller pieces’. Often, special photographic systems are employed to record the fluorescent image of DNA bands in electrophoresis gels. These photographic systems are expensive and are generally dedicated solely to photographing ethidium-stained DNA in electrophoresis gels. Though UV light devices and photographic systems are part of the basic equipment in most molecular biology laboratories, they are expensive, require space, limit the environment in which molecular genetics can be applied, and impede the use of basic molecular biology techniques in undergraduate studies. A more serious drawback to the use of ethidium bromide for visualizing DNA in electrophoretic gels is its toxicity. Ethidium bromide is a mutagen’ and must therefore be used with care. This danger re0

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stricts the use of ethidium bromide to laboratories with well disciplined and experienced scientific personnel. Now that there is interest in teaching molecular biology in secondary schools, a safer technique for visualizing DNA in electrophoresis gels is needed. Chemiluminescence seems ideally suited for secondary school laboratories owing to its safety, convenience and low cost.

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Gel preparation

Using chemiluminescence to visualize DNA in agarose gels In this report we describe a new chemiluminescent detection technique for visualizing DNA in electrophoresis gels. This technique is simple, sensitive, and requires only an electrophoresis system and photographic film. The new method is illustrated by imaging electrophoretically separated bands of biotinylated, Hind III Lambda DNA in agarose gels. The chemiluminescent system employed to label and illuminate the bands of biotinylated DNA (B-DNA) include luminol and streptavidin conjugated to horseradish peroxidase (SA-HRP). With a chemiluminescent enhancer, 10 to 20 pg per band of B-DNA are routinely detected. This is nearly a lOOO-fold increase over that obtained by ethidium bromide staining.

The gel is reduced in thickness by dehydration. This may be accomplished quite economically by blotting with paper towels. Filter paper (Whatman No. 1) is used to cover both surfaces of the electrophoresis gel. The filter paper and gel sandwich is placed between layers of paper towels to absorb most of the water from the gel. A small weight (approximately 500 g) is placed on top to press the gel firmly against the paper towels. The weight also helps to prevent the gel from wrinkling and curling once it begins to dry. Though the gel is dry enough for use in a few hours, we generally dry overnight for convenience. A commercial gel drier can also be used to dehydrate the gel. Though a gel dryer is more expensive than paper towels, it reduces drying time to less than one hour. This time saving device makes it possible to visualize electrophoretically separated bands of B-DNA in less than one day. Both surfaces of the gel are covered with filter paper (Whatman No. l), sandwiched between cellophane, placed on the gel drier and covered with a rubber sheet. A vacuum of 25 to 29 inches of Hg is applied and the dryer is set to 50°C. Drying times of 30 to 40 min have been used with success.

Sample preparation

Labeling and visualizing B-DNA chemiluminescent-

Of the many techniques for biotinylating DNA3, an easy and straightforward approach employs photoactivated biotin4. This labeling reagent is available commercially from many suppliers. Biotinylated DNA is prepared by adding 100 yl of photobiotin (Vector Laboratories, Burlingame, CA, U.S.A) to lOO@ of a 1 pug/ml solution of Hind III Lambda DNA and irradiating with visible light for 15 min under a 150 W light bulb (Sylvania DWC 150 W Flood Light) placed 10 cm directly above the sample. The reactants are stored frozen as a stock solution of B-DNA without further purification.

1Y

Whether B-DNA in agarose gels is labeled before or after the gel is dried, the protocol for labeling is the same. A mini gel of agarose (5 x 7.5 cm) is soaked in deionized water for 10 min, then labeled for 60 min in 40 ml of labeling solution (100 mM Tris-150 mM NaCl, pH 8.5 with 0.25 pg SA-HRP/ ml). Unbound SA-HRP is removed from the gel by washing with agitation. The gel is washed three times for 10 to 15 min per wash with 100 mM Tris-150 mM NaCl, pH 8.5 solution containing 100~1 of Tween 20 per liter. If the electrophoresis gel has not been dried prior to labeling with SA-HRP, the drying procedure

Electrophoresis

A portion of the stock B-DNA solution is diluted with deionized water (18 MQ) to concentrations ranging from 0.01 to 100 ng/pl. The dilutions are heated for 5 min at 65°C cooled on ice for 1 min, and then loaded onto an electrophoresis 0.8% agarose slab gel. B-DNA is electrophoresed in a solution of Tris-borate electrophoresis buffer’ using a mini-gel electrophoresis cell (IBI, New Haven CT, U.S.A.) at 10 V/cm for 2 h. After electrophoresis, the gel can be processed to visualize chemiluminescent bands of DNA or it can be wrapped in plastic wrap and stored in the refrigerator for visualization several days later.

Fig. 1. Contact print apparatus. A = Black plastic cover, B = shutter, C = clear plastic sheet, D = dehydrated electrophoresis gel, E = Polaroid film pack, F = Polaroid film pack holder.

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described above is used. Once the gel is dried, labeled with SA-HRP and washed, chemiluminescent reagent is added and the luminescent bands of BDNA are recorded on film. Chemiluminescent

then be followed by a shorter or longer exposure depending on the results obtained with the first photograph. We find the simple photographic system described above quite flexible. It allows us to obtain both short (less than 1 s) and long exposures (hours).

reagent

A stock solution of luminol is prepared by dissolving 10 mg of 5-amino-2,3-dihydro-1,4_phthalazinedione (Sigma) in 1 ml of dimethylsulfoxide. This solution is stable for at least one month if stored refrigerated. Working chemiluminescent reagent is prepared by dissolving 15 ~1 of stock luminol reagent into 2 ml of 100 mM Tris, pH 8.0. Rapid stirring on a vortex mixer is necessary to ensure that the luminol dissolves in the aqueous buffer. A pH of 8.0 was used to provide a chemiluminescent signal-to-noise ratio acceptable for high speed photographic film. If the chemiluminescent signal were processed electronically, perhaps a higher pH buffer could be used to increase sensitivity. A volume of 5 ,~l of a 3% hydrogen peroxide solution is added to the luminol, Tris buffered solution. In our hands this reagent remains stable for at least one day at room temperature. If picograms of DNA per band are to be visualized, an enhancer such as p-phenylphenol (PPP) or p-coumaric acid (PCA) must be used5. We routinely use concentrations of 24 ,LLMPPP or PCA luminol-based chemiluminescent reagent with good results. Photographing the chemiluminescent B-DNA

Fig. 1 shows how the SA-HRP labeled gel is placed onto the stage of a Polaroid film pack holder (Polaroid MP-4, No. 44-48). A clear polyethylene sheet cut from a food storage bag is placed over the shutter opening of the Polaroid film pack holder. The labeled gel is then centered on top of the plastic sheet. Approximately 2 ml of working luminol reagent is poured over the gel. The gel is allowed to stand in the working chemiluminescent reagent for 5 min. This allows the reagent to soak into the gel and also helps to avoid overexposing the film by a flash of luminescence. Excess working chemiluminescent reagent is removed by placing an adsorbent paper next to the gel. Once the pool of luminescent reagent is removed, the gel is covered with a second piece of plastic film. To expose the film, we move the gel and film pack holder to a room where the light from windows is blocked and light fixtures can be turned off. Darkroom conditions, however, are not needed. To expose the Polaroid film to the chemiluminescent image of B-DNA in the electrophoresis gel, the camera and gel are covered with two layers of black plastic and the shutter is opened. When there is uncertainty regarding the amount of luminescence the gel will emit, a 30-s exposure is recommended. This can

Polaroid instant film

Polaroid 107 film (ASA 3000) is best for photographing chemiluminescent images in gels. Though higher speed Polaroid 612 film (ASA 20 000) is adequate for this work, it does not possess the contrast provided by slower speed film. Better resolution of individual bands with a broader range of DNA mass is an important advantage provided by higher contrast film. Depending on the amount of DNA loaded into the gel and on whether enhanced or standard working chemiluminescent reagent is employed, exposure time can range from 5 s to 60 min. Because the SA-HRP and luminol chemiluminescent system provides strong chemiluminescence rapidly, photographs of the luminescent DNA can be obtained within a short time (usually several minutes). Fig. 2 shows the chemiluminescent image of Hind III, Lambda DNA bands separated electrophoretitally in an agarose gel. In Fig. 2B the six major bands of Hind III Lambda DNA can be seen in lanes A-C. The smallest amount of DNA detected is in lane E band 3. Assuming no loss of DNA during loading of the gel, this band contains approximately 1.1 pg of B-DNA.

B

A

LANES

LANES

ABCDE

85 24 8

5 6

6

Fig. 2. Chemiluminescent DNA in an agarose electrophoresis gel. Different amounts of biotinylated Hind III Lambda DNA were loaded into the gel: lane A, 5 ng; lane B, 1 ng; lane C, 200pg; lane D, 4Opg; and lane E, 8pg. After electrophoresing, proces ing and adding enhanced chemiluminescent reagent, photographic exposures were made for (A) 30 s, and (B) 30 min.

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Using X-ray film

Photographic sytems employed for autoradiography can be used for capturing luminescent images of DNA in agarose electrophoretic gels. Using Kodak X-Omatic film we have obtained excellent photographs of luminous bands of DNA in agarose gels. Though exposure time must be increased from 1.5 to 2 times that employed with Polaroid 107 film, XOmatic film provides slightly better contrast, making it easier to see low levels of luminescence.

Acknowledgements We are grateful to Dr. Douglas Vizard, Director of Research at International Biotechnology, Incorporated, New Haven, Connecticut, for providing us with Kodak X-Omatic film and for developing our exposures. This work was funded by a DOE SBIR Contract number DE-ACOl-87ER80468.

References Conclusion Because of its safety, convenience and sensitivity, the development of chemiluminescence-based technology is an important goal. Imaging DNA in electrophoresis gels is but one example of how chemiluminescent technology can be employed. Armed with little more than a Polaroid film pack holder, film and an electrophoresis unit, it is now possible to do restriction mapping of DNA. Furthermore, this work need not take great quantities of DNA. The ease, simplicity, safety and economy of this chemiluminescent technique makes it an excellent procedure for experienced molecular biologists, in addition to those learning the basic skills of DNA analysis.

T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982. M. J. Waring, Biochim. Biophys. Acta, 114 (1966) 243. J. L. McInnes, S. Dalton, P. D. Vize and A. J. Robins, Biol Technology, 5 (1987) 269. A. C. Foster, J. L. McInnes, D. C. Skingle and R. H. Symons, Nucleic Acid Res., 13 (1985) 745.

H. G. Thorpe (1986) 331.

and L. J. Kricka,

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Margaret E. &person is a research scientist at SymBiotech, Inc., 8 Fairfield Blvd., Wallingford, CT 06492, U.S.A. Robert W. Coughlin is Vice President of SymBiotech, Inc., Wallingford, CT, U.S.A., and a Professor of Chemistry and Chemical Engineering at the University of Connecticut, Storrs, CT, U.S.A. Edward M. Davis is President of SymBiotech, Inc.

book reviews A computer-assisted

LC system

A Computer-Assisted Chromatography System, by K. Jinno, Hiithig Verlag, Heidelberg, 1990, DM 118.00, US$70.00 (v + 27lpages), ISBN: 3-7785-1497-O This book is mainly a summary of the work done by Jinno which led to a microcomputer-assisted system, McASSYST, for liquid chromatography. In fact, chapter 6 (nearly 60 pages) consists of a set of BASIC programs related to the system. Nearly all examples deal with the separation and identification of polycyclic aromatic hydrocarbons (PAHs). The wealth of information about this class of substances is such that scientists interested in the analysis of these compounds should buy this book. Chapter 1 is a short introduction to the book and Chapter 2 describes a

database for literature data on HPLC for PAHs. Chapter 3 is the longest and also the more interesting chapter, consisting essentially of a QSAR (quantitative structure-activity relationship) approach to describing chromatographic behaviour of PAHs and, more briefly, of smaller classes of compounds. The retention behaviour of PAHs is first related to descriptors such as log P, a measure of hydrophobicity that can be predicted using additive rules based on structural fragments, and also to descriptors such as length/breadth related to the shape of the molecule. In a second step, logk’is also related to the percentage of organic modifier (methanol or acetonitrile). This is described as an optimisation. In fact, the author tries to predict a composition of the mobile phase such that the reten-

tion and separation obey criteria which are given by the user. This approach seems to be successful for PAHs but it is to be expected that it is not generally useful, i.e. not necessarily for other substances. Indeed, the author optimizes retention but not selectivity. The use of ternary phases, which is sometimes so successful, is never tried. The author states that optimization can be carried out by methods such as window diagrams (very useful in GC, but much less in HPLC) or Simplex but he never mentions mixture designs such as the solvent triangle or other factorial designs. The methods proposed should only work well with neutral compounds since pH is never considered, the predicted separations are based on Gaussian peak assumptions and no tailing suppressor is added when bases are eluted. Another drawback of the approach used by the author is that it requires a lot of data. Indeed, to obtain the rela-