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Improved resolution of DNA fragments in polysaccharide-supplemented agarose gels
ANALYTICAL
BIOCHEMISTRY
Improved
163,247-254
Resolution
DANIELPERLMAN,
(1987)
of DNA Fragments in Polysaccharide-Supplemented Agarose Gels HEMANT~HIKARMANE,
AND HARLYNO.HALVORSON
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254 Received October 20, 1986 The electrophoretic separation of nucleic acids, including small DNA fragments in the range 50- 1000 bp, is presently carried out in polyacrylamide gels or in gels containing high concentrations of agarose. We have developed an alternative gel matrix composition which is inexpensive, nontoxic, easy to prepare, and highly transparent to visible and uv light. The composition combines a soluble nonionic polysaccharide such as hydroxyethylcellufose, metbylcellulose, or galactomannan with a minimum but sufficient concentration of agarose to form a gel which immobilizes the “liquid phase sieve.” These mixtures do not replace polyacrylamide for resolving fragments smaller than approximately 75 nucleotides. However, the new gels show DNA fragment resolution (band separation versus distance traveled) and optical clarity superior to those of conventional agarose. Q 1987 Academic PIW, Inc. KEY WORDS: DNA electrophoresis; soluble cellulose; molecular sieving; agarose gels.
Two molecular sieving agents used in gel electrophoretic fractionation of nucleic acids and proteins are polyacrylamide and agarose. Because of its smaller pore size, polyacrylamide is generally more effective in separating small molecules. In improving the ability of agarose to resolve small DNA fragments, chemically modified agaroses with reduced pore size have been developed. Hydroxyethylation of agarose, for example, reduces average pore diameter approximately 50%, resulting in increased sieving and slower migration of molecules (1). This modification simultaneously decreases intermolecular associations in the agarose matrix, resulting in the well-known “low melting” agarose commercial products. Various soluble polymers have also been added to agarose to modify its physical, chemical, and electrical properties. For example, a variety of soluble nonionic polymers have been added to decrease electroendosmosis and to improve the quality of isoelectric focusing (2) and electroimmunodiffusion in agarose gels (3,4). Hydrocolloid polymers have also been added to facilitate rehydration of dried gels
(5). Polyacrylamide may also be added to agarose gels as a soluble uncrosslinked solution (6) or polymerized and crosslinked with bisacrylamide in situ (7). It has been recently shown that an uncrosslinked polyacrylamide solution is capable of molecular sieving even in the absence of agarose (8). Disadvantages encountered in utilizing acrylamide include gel preparation time, expense of pure acrylamide, chemical toxicity, and susceptibility of solutions to air oxidation. Therefore, we investigated a number of other soluble nonionic polymers of natural and synthetic origin which might be added to agarose to improve its ability to sieve small DNA fragments. A number of polysaccharides, including soluble celluloses, galactomannan, starch, dextran, and Ficoll, were tested. We now report that certain cellulose and galactomannan solutions can form molecular sieves for electrophoresis. As liquid phase sieves, the resolution of molecular species is significantly compromised due to solution mixing. However, when combined with low concentrations of agarose, the resulting com-
247
0003-2697187 $3.00 Copyright 0 1987 by Academic F’ress, Inc. All rights of reproduction in any form reserved
248
PERLMAN,
CHIKARMANE,
posite gels are superior to high-concentration agarose gels in their ability to fractionate small DNA fragments. The composite gels are inexpensive, easily poured from a melt, and have excellent uv and visible light transparency. MATERIALS
AND
METHODS
Electrophoretic conditions. Tris-borate EDTA (TBE)’ buffer (9) containing ethidium bromide (1 &ml) was used as the electrophoresis buffer in all experiments. Minigels were poured on 75 X 50-mm glass slides which held 10 ml of gel. Samples were loaded in 5% glycerol containing 0.05% each of xylene cyanole FF and bromphenol blue as marker dyes and electrophoresed at 5 V/cm. Preparation of soluble cellulose solution. HEC was prepared as a 2% (w/v) stock solution in distilled water by stirring continuously until the solution became clear and very viscous, after which it was left overnight to ensure homogeneity. NaN3 (0.1%) was added as a bacteriostatic agent. Solution aliquots were weighed rather than pipetted due to high viscosity. Preparation of composite gels. (a) For HEC-, MC-, and GM-agarose gels, hot aqueous agarose was mixed with appropriate volumes of hot polysaccharide solution and TBE buffer. Gels were poured after bubbles had floated to the surface. (b) Amylose, Fico11 400, and dextran-agarose gels were prepared by dissolving the appropriate powder directly into hot agarose. Preparation of sectored gels. Sectored minigels were prepared by inserting, for example, two evenly spaced acrylic dividers between the teeth of the sample well-forming comb. The outer sectors were poured first, and after removal of the dividers, the center section was poured. Tube gel electrophoresis. Quartz tubing was used to allow direct uv photography of ’ Abbreviations MC, methylcellulose; borate EDTA.
used: HEC, hydroxyethylcellulos; GM, galactomannan; TBE,
Tris-
AND
HALVORSON
ethidium bromide-stained DNA within the tube. Grade 5 HEC was mixed with 0.1 vol of 10X TBE containing ethidium bromide and drawn into the tube. The bottom was sealed with dialysis tubing and an elastic band. The membrane ensured electrical conductivity without loss of HEC. As an alternative, a rectangular agarose frame (0.7% agarose in TBE) was formed on uv-transparent plastic. This frame contained the sample wells and held soluble polymers used for DNA fractionation. Buffer in the horizontal chamber maintained electrical contact with the agarose but did not flow over it. After electrophoresis, the uv-transparent plate was placed directly on the uv box for photography. HEC viscosities. The viscosities of the various grades of HEC at defined w/v concentrations are grade 1, 7.5-l 50 cp at 5%; grade 2, 150-400 cp at 2%; grade 3, 1500-2500 cp at 2%; grade 4, 4500-6500 cp at 2%; and grade 5, 3400-5000 cp at 1% solution. Chemicals. Agarose (type ME), HEC, and GM (isolated from locust bean gum) were from the FMC Corp. Ficoll 400 was from Pharmacia. Dextran @&, 5-40 X 106) and MC were from Sigma Chemicals. DNA restriction fragment size standards were from New England Biolabs. RESULTS
Molecular Sieving of DNA in Cellulose Solutions After making the preliminary observation that the addition of HEC to agarose gels enhances the resolution of small DNA fragments, we investigated the possibility that HEC itself had intrinsic sieving capability. DNA restriction fragments ranging from approximately 70 to 1400 bp were applied to the tops of quartz tubes holding HEC solutions and were electrophoresed vertically. Grade 5 HEC (having maximum intrinsic viscosity) was used to minimize diffusion-related mixing during solution-phase electrophoresis. DNA fragments were partially resolved in this medium despite some electro-
IMPROVED
ELECTROPHORETIC
phoretic coning artifacts (Fig. 1, gel a). A technically better result was obtained with horizontal electrophoresis in DNA in HEC solution contained within an agarose frame (Fig. 1, gel b). The best resolution (Fig. 1) in this liquid sieve was obtained for DNA fragments smaller than 600 bp (1.5% HEC) or 400 bp (2.0% HEC). Methylcellulose and galactomannan acted as comparable molecular sieves in liquid phase; solutions containing as little as 0.5% (w/v) galactomannan were effective in fractionating DNA fragments smaller than 70 bp (data not shown). These experiments showed that a number of nonionic polysaccharide solutions had intrinsic DNA-sieving ability. However, the practical use of such nongelled sieving matrices is limited due to diffusion and convection artifacts.
Addition of HEC to agarose gels improves the sieving of small DNA fragments. Agarose and HEC were mixed to form composite gels since reduction of diffusion-related artifacts inherent in the liquid HEC sieving medium was desirable. To compare sieving in a composite gel with that in a simple agarose gel having a similar “solids” content, a sectored gel was utilized, with DNA being simulta-
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DNA FRACTIONATION
neously electrophoresed in gel sectors containing either 0.7% agarose with 1.3% HEC (grade 4) or simple 2.0% agarose. The addition of HEC clearly improved the sieving potential of agarose in the separation of small DNA fragments (Fig. 2, gels a and b). Small DNA fragments were better resolved in the HEC-agarose gel (Fig. 2). The break in the HEC-agarose curve around 600 bp is consistent with that observed in Fig. 1, demonstrating the strong influence of HEC on the fractionation behavior of the composite gel. The excellent linearity maintained at the small molecular size end of the HEC-agarose plot (and poor linearity for agarose) suggests that HEC solutions have a smaller “pore size” than conventional agarose gels. Resolution of DNA fragments larger than 1000 bp appeared to be comparable in the two gels (data not shown).
Sieving ejiciency is a function of HEC concentration. If HEC were primarily responsible for DNA sieving in HEC-agarose gels, then varying the agarose concentration at a fixed HEC level should have little effect on DNA fragment mobility. This prediction was confirmed in experiments varying agarose content between 0.4 and 1.6% (w/v)
a
b
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FIG. 1. Electrophoresis of DNA in HEC solutions. HueIII-cleaved 4X174 RF DNA (150 ng) was electrophoresed (gel a) in submerged vertical quartz tubes (3-mm id. X 15-cm length) containing (w/v) 1.5% (left) or 2.0% (right) HEC, grade 5, dissolved in buffer containing ethidium bromide (2 pg/ml). Mobilities of the electrophoresed fragments are plotted: 1.5% HEC (a), 2.0% HEC (A). $X174 RF DNA fragments (gel b, left) and Mspkleaved pBR322 DNA (gel b, right) were electrophoresed in a 2% HEC solution (grade 5) contained in a horizontal agarose frame.
250
PERLMAN,
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CHIKARMANE,
AND HALVORSON
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FIG. 2. Electrophoresis of DNA in HEC-agarose and GM-agarose composite gels. A two-part gel was prepared containing 2.0% agarose (a) and 1.3% HEC (grade 4) plus 0.7% agarose (b). The minigel (&cm length) was submerged in buffer and preelectrophoresed for 20 min (5 V/cm) before the sample DNAs were loaded. HueIII-cleaved dX I74 RF DNA (lanes 1 and 3) and MqI-cleaved pBR322 DNA (lanes 2 and 4) (200 ng each) were applied to the gel. Plots of DNA mobility include both &Xl74 and pBR322 restriction fragments: agarose gel (0); HEC-agarose gel (A). Another two-part gel was prepared containing 2.0% agarose (c) and 0.7% agarose plus 0.5% GM (d). DNA markers and electrophoresis were as described above except that lanes 1 and 3 contained a mixture of HaeIII-cleaved @X 174 RF DNA and MS&cleaved pBR322 DNA fragments.
with HEC fixed at 1.0% (data not shown). Subsequently, with agarose concentration held constant at 0.7%, we varied the HEC concentration and monitored DNA sieving ability (Fig. 3A). At the lowest concentration of HEC, 0. l%, there was only minor retardation of small DNA fragments but with increasing HEC levels, DNA fragment mobility steadily decreased relative to fragment migration rates in conventional agarose. The accompanying change in the slopes of the log molecular size vs mobility curves reflects improvement in sieving efficiency. Normalizing these curves to a common migration distance accentuated the difference in slopes (and hence resolution). The changing y-coordinate position of the breakpoint in the curves in Fig. 3A establishes the maximum DNA fragment size which can be optimally fractionated using a given concentration of HEC. This position,
which decreased as the HEC concentration increased, may represent an “exclusion point” at which pore size in the HEC-contaming matrix becomes limiting. Above this point, sieving may more closely resemble the original agarose gel. The upper limit to the concentration of HEC which may be added to agarose gels is determined by solubility [approximately 2% (w/v)]. For 0.5-0.7% (w/v) agarose gels, addition of more than 2-2.5% HEC produced a cloudy granular gel having inferior sieving properties.
Sieving eficiency improves with increasing intrinsic viscosity of HEC. Several observations were made by comparing HEC-agarose composite gels containing HEC of differing viscosities. (i) Gel strength as determined by resistance to gel collapse (for 0.2% agarose1.O% HEC gels subjected to 50,OOOg centrifugation for 30 min) was much greater with high- than with low-viscosity HEC. The per-
IMPROVED
ELECTROPHORETIC
centage volume collapse for a gel containing grade 1 HEC was 59% vs only 3 and I%, respectively, for HEC grades 3 and 4. Supernatants liberated from these partially collapsed gels contained the original concentration of HEC (l.O%, w/v), suggesting that HEC was rather loosely held within the aga-
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FIG. 3. Effect of increasing HEC concentration and HEC viscosity on DNA sieving. A four-part gel was prepared containing 0.7% agarose with differing concentrations of HEC (viscosity grade 4) in each part. To prevent HEC leaching, the minigel (5cm length) was covered with plastic wrap prior to submerging in buffer. HindIII-cleaved phage X DNA and HueIII-cleaved &Xl74 RF DNA (200 ng each) were applied to the gel. (A) The relative electrophoretic mobilities of DNA fragments are plotted against log of molecular size. sectors of gel contained unsupplemented agarose (0) and agarose plus 0.1% (0), 0.3% (+), and 1.0‘76 (A) (w/v) HEC. (B) A four-part gel was prepared as above except that the gel sectors contained 0.5% agarose plus 1.5% HEC of differing intrinsic viscosities. HueIII-cleaved $X I74 RF DNA (200 ng) was electrophoresed in gel lanes containing HEC viscosity grade 1 (O), grade 2 (0), grade 3 (+), and grade 4 (A).
DNA FRACTIONATION
251
rose gel matrix; i.e., the affinity of HEC for water was greater than that for the agarose matrix. (ii) Loss of HEC from submerged gels to the surrounding buffer (termed leaching) was measurable with all HEC viscosities tested. Leaching occurred more rapidly with low- than with high-viscosity HEC and explained why preelectrophoresis was sometimes required to stabilize the electrophoretic properties of submerged HEC-agarose gels. However, HEC leaching was prevented and preelectrophoresis was obviated by covering the gels (except for the sample wells) with a microscope slide or plastic wrap prior to submerging. Given the above observations showing physical properties of agarose gels varying with HEC viscosity, we studied DNA sieving as a function of HEC viscosity. A sectored gel containing 0.5% agarose and 1.5% HEC of varying viscosity (grades 1, 2, 3, and 4) was prepared. The gel was covered with a glass slide, submerged in buffer, loaded with DNA, and electrophoresed. The higher the HEC viscosity, the better the separation of DNA fragments (Fig. 3B). The low-intrinsicviscosity grade 1 HEC did not sufficiently improve the sieving properties of conventional agarose to be considered useful. Based on these experiments adequate intrinsic viscosity is believed to be a prerequisite for effective molecular sieving by soluble polymers. Other Carbohydrate Polymers May Have a Positive, Neutral, or Negative Efect on DNA Fractionation by Agarose Having demonstrated that soluble cellulose derivatives were capable of DNA sieving, we investigated the sieving properties of other nonionic carbohydrate polymers. As examples, four polymers having high molecular weights but differing in physical structure were chosen. GM is a polymer of #?l-4linked mannose residues with single a-D-galactosyl side chains. Ficoll 400 (-400,000 Da), is a synthetic copolymer of sucrose and epichlorohydrin and has a branched struc-
252
PERLMAN,
CHIKARMANE,
ture. Dextran (5-40 X 10” Da) is a linear polymer consisting mainly of LY-1,6-linked glucose residues. Soluble starch (amylose) is an cr l-4-linked glucose polymer and has a helical structure. For each polymer, sectored agarose gels were prepared with the sectors containing differing concentrations of the above polymers (between 0 and 4.0%, w/v). Initially, DNA fragment separation was monitored in composite gels containing GM. $X174 RF and pBR322 DNA fragments were electrophoresed in 0.7% agarose containing 0.5% GM and in a simple 2.0% agarose gel (Fig. 2, gels d and c, respectively). The significant contribution to molecular sieving and the increase in resolution produced by GM were especially pronounced for DNA fragments larger than 200 bp (cf. top six restriction fragments, lanes 2 and 4, Fig. 2, gels c and d). Increasing the GM concentration above 0.5% should further increase the resolution of fragments ~200 bp in a manner analogous to HEC (Fig. 3A). However, due to the rubbery consistency and high gelling temperature of GM-agarose solutions, practical use of such higher GM concentrations would probably require partial hydrolysis of either the GM or the agarose. The minimal amount of GM leaching from GM-agarose gels, attributable to strong intermolecular association (lo), obviated measures to prevent leaching which occurred with submerged HEC-agarose gels. With regard to the other polymers, electrophoresis in agarose-containing dextran or Fico11 of DNA fragments ranging in size from 0.10 to 23 kb showed essentially no alteration in DNA migration rates (data not shown). This constancy was maintained despite significant changes in sol viscosity apparent before gel formation and in gel strength and elasticity resulting from the mixed compositions. Amylose-containing agarose gels, however, exhibited a remarkably altered electrophoretic sieving “phenotype.” While DNA fragments larger than approximately 4000 bp migrated at comparable rates in gels with and without starch, smaller fragments were
AND
HALVORSON
significantly retarded by the starch. A great range of DNA fragment sizes can be simultaneously displayed in a single amylose-agarose gel (Fig. 4). This electrophoretic “compression,” which is analogous to that produced by a gradient gel composition, is essentially opposite to the effect produced by HEC (Fig. 3A) and may be useful in surveying or comparing restriction digestion patterns from large DNA segments. We suggest that the selective retardation of small DNA fragments in amylose-agarose represents some type of size-exclusion process. If, in contrast, the retardation were caused by amylose binding to DNA, then we would expect all DNA fragment mobilities to be proportionately affected. DISCUSSION
A number of nonionic soluble polymers have been shown to sieve DNA molecules during electrophoresis. These polymers, which include soluble cellulose derivatives and galactomannan, can replace a substantial proportion of the agarose in conventional electrophoretic gels. Thus, a 1% HEC solution solidified by a minimum but sufficient concentration of agarose, e.g., approximately 0.4%, can replace a 2.0% agarose gel. A comparison of HEC-agarose and conventional agarose-sieving characteristics for small DNA fragments suggests that the effective pore size is smaller in the HEC network than in the agarose matrix. This reduction in pore size leads to increased resolution of DNA fragments as well as to improved linearity in the plot of log base pairs vs mobility, especially in the lower size range (see Fig. 2). Other advantages of HEC-containing gels over conventional agarose gels are (i) improved transparency with both uv and visible light, (ii) lower material cost, and (iii) reduced electrophoretic run time. We have produced rapidly dissolving dry mixtures of agarose and soluble cellulose which will soon be commercially available. Fractionated macromolecules are easily eluted and precipitated from HEC-agarose
IMPROVED
ELECTROPHORETIC
gels for analytical and preparative purposes. For example, HEC remains soluble in aqueous solutions containing >70% ethanol and does not interfere with the precipitation and subsequent enzymatic treatment of DNA restriction fragments. Thus we have recovered DNA restriction fragments having 5’ protruding ends from HEC-agarose gels treated with DNA polymerase Klenow enzyme to produce blunt ends and successfully ligated this DNA with cleaved pBR322 DNA. Plasmid DNA fragments similarly purified have been nick-translated, producing hybridization probes of specific activity equal to that of DNA purified from conventional agarose gels. We have additionally shown that DNA fragments in HEC-agarose gels can be electrophoretically transferred to nylon and nitrocellulose membranes (unpublished results). Besides increasing the sieving ability of agarose gels, cellulose derivatives greatly in-
3x104
crease the viscosity of the aqueous phase within the gel. We believe that this increased viscosity and the reduction in free water volume within the gel (caused by the hydrophilic polymer) contribute to the high-resolution sharp DNA bands observed in these gels. Other polymers including amylose produced similar DNA band “tightening” yet, together with other nonionic polymers such as Ficoll and high-molecular-weight high-viscosity dextran, did not assist in sieving DNA. Simple low-molecular-weight viscositybuilding agents did not appear to help the sieving process either. For example, we have observed that a 20% sucrose solution added to an agarose gel greatly reduced the rate of DNA electrophoresis. However, all of the DNA fragments were proportionately retarded. With regard to HEC-agarose composite gels, we know that agarose is not required for DNA sieving but minimizes physical mixing
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DNA FRACTIONATION
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FIG. 4. DNA sieving in amylose-agarose gels. A three-part gel was prepared containing 0.7% agarose with differing concentrations of amylose. The gel was protected against leaching and the marker DNAs were applied as described in Fig. 3A. Data for agarose (0) [gel (a)], agarose plus 2% amylose (0) [gel (b)], and agarose plus 4% amylose (+) are provided. Lanes 1 and 3, &X174 RF DNA; lanes 2 and 4, phage X DNA.
254
PERLMAN,
CHIKARMANE,
within the HEC [cf. Fig. 2 (with agarose) and Fig. 1 (without agarose)]. The weakness of physical and chemical interactions between HEC and agarose was suggested by two direct observations: (i) the concentration of HEC material in supematants of centrifuged composite gels was the same as that originally included in the gels, and (ii) the gelling and remelting temperatures for 0.7% agarose in TBE buffer (35 and 85”C, respectively) were not measurably altered by HEC addition. The physical mechanism by which soluble cellulose or galactomannan interacts with DNA to promote sieving is still unknown. It is possible that helical secondary and/or tertiary structures of carbohydrate polymers play a role in the sieving process. This is deduced from the observations that Ficoll and dextran, which both lack defined secondary and tertiary structures, failed to affect DNA sieving in agarose gels. Helical structures would have considerably more rigidity in solution than random polymers in forming a sieving matrix. Do polysaccharide helices form a systematic noncovalent network of molecules in solution? One clue is that the efficient sieving of DNA occurring in highviscosity HEC solutions was lost with lowviscosity HEC. This sieving characteristic did not change with the addition of a heterologous physical matrix, i.e., the agarose gel used to immobilize the HEC solutions. Therefore we believe that an extended network must exist among HEC molecules themselves for sieving to occur. Furthermore, noncovalent helix-helix binding between soluble polysaccharides, leading to gel formation, has already been demonstrated for agarose and galactomannan ( 10) and also xanthan and galactomannan (11). It is possible that an analogous though weaker binding occurs between agarose and HEC based on
AND HALVORSON
our studies on leaching of HEC from agarose. Curiously, both HEC and galactomannan which have 01-4 linkages increase agarose sieving efficiency, while amylose, with (Yl-4 linkages, decreases the efficiency. All of these polymers including agarose itself (12) have helical structures which appear necessary but apparently are not sufficient for polysaccharides to form sieving matrices. ACKNOWLEDGMENTS We acknowledge support from NIH Grant GM 18904 (D.P.) and from International Minerals & Chemicals Corporation, Northbrook, IL (H.C.). We thank the FMC Corp., Marine Colloids Division, for experimental material and for support in publishing this work and Dr. Hillel Levinson for his review of the manuscript.
REFERENCES 1. Cook, R. B. (1982) U.S. Patent No. 4,319,975. 2. Cook, R. B., and Witt, H. J. (1981) U.S. Patent No. 4,290,9 11. 3. &huller, E., Lefevre, M., and Tompe, L. (1972) Clin. Chim. Acta 42, S- 13. 4. Johansson, B. G., and Hjerten, S. (1974) Anal. Biothem. 59,200-2 13. 5. Renn, D. W., and Mueller, G. P. ( 1970) U.S. Patent No. 3,527,7 12. 6. Bode, H.-J. (1977) Anal. Biochem. 83,204-2 10. 7. Peacock, A. C., and Dingman, C. W. (1968) Biochemistry I, 668-674. 8. Tietz, D., Gottlieb, M. H., Fawcett, J. S., and Chrambach, A. (1986) Electrophoresis 7, 217-220. 9. Maniatis, T., Fritsch, E. F., and Sambrook, J. ( 1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 10. Dea, I. C. M., McKinnon, A. A., and Rees, D. A. (1972) J. Mol. Biol. 68, 153-172. 11. Cairns, P., Miles, M. J., and Morris, V. J. (1986) Nature (London) 322,89-90. 12. Amott, S., Fulmer, A., Scott, W. E., Dea, I. C. M., Moorhouse, R., and Rees, D. A. (1974) J. Mol. Biol. 90,269-284.
BIOCHEMISTRY
Improved
163,247-254
Resolution
DANIELPERLMAN,
(1987)
of DNA Fragments in Polysaccharide-Supplemented Agarose Gels HEMANT~HIKARMANE,
AND HARLYNO.HALVORSON
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254 Received October 20, 1986 The electrophoretic separation of nucleic acids, including small DNA fragments in the range 50- 1000 bp, is presently carried out in polyacrylamide gels or in gels containing high concentrations of agarose. We have developed an alternative gel matrix composition which is inexpensive, nontoxic, easy to prepare, and highly transparent to visible and uv light. The composition combines a soluble nonionic polysaccharide such as hydroxyethylcellufose, metbylcellulose, or galactomannan with a minimum but sufficient concentration of agarose to form a gel which immobilizes the “liquid phase sieve.” These mixtures do not replace polyacrylamide for resolving fragments smaller than approximately 75 nucleotides. However, the new gels show DNA fragment resolution (band separation versus distance traveled) and optical clarity superior to those of conventional agarose. Q 1987 Academic PIW, Inc. KEY WORDS: DNA electrophoresis; soluble cellulose; molecular sieving; agarose gels.
Two molecular sieving agents used in gel electrophoretic fractionation of nucleic acids and proteins are polyacrylamide and agarose. Because of its smaller pore size, polyacrylamide is generally more effective in separating small molecules. In improving the ability of agarose to resolve small DNA fragments, chemically modified agaroses with reduced pore size have been developed. Hydroxyethylation of agarose, for example, reduces average pore diameter approximately 50%, resulting in increased sieving and slower migration of molecules (1). This modification simultaneously decreases intermolecular associations in the agarose matrix, resulting in the well-known “low melting” agarose commercial products. Various soluble polymers have also been added to agarose to modify its physical, chemical, and electrical properties. For example, a variety of soluble nonionic polymers have been added to decrease electroendosmosis and to improve the quality of isoelectric focusing (2) and electroimmunodiffusion in agarose gels (3,4). Hydrocolloid polymers have also been added to facilitate rehydration of dried gels
(5). Polyacrylamide may also be added to agarose gels as a soluble uncrosslinked solution (6) or polymerized and crosslinked with bisacrylamide in situ (7). It has been recently shown that an uncrosslinked polyacrylamide solution is capable of molecular sieving even in the absence of agarose (8). Disadvantages encountered in utilizing acrylamide include gel preparation time, expense of pure acrylamide, chemical toxicity, and susceptibility of solutions to air oxidation. Therefore, we investigated a number of other soluble nonionic polymers of natural and synthetic origin which might be added to agarose to improve its ability to sieve small DNA fragments. A number of polysaccharides, including soluble celluloses, galactomannan, starch, dextran, and Ficoll, were tested. We now report that certain cellulose and galactomannan solutions can form molecular sieves for electrophoresis. As liquid phase sieves, the resolution of molecular species is significantly compromised due to solution mixing. However, when combined with low concentrations of agarose, the resulting com-
247
0003-2697187 $3.00 Copyright 0 1987 by Academic F’ress, Inc. All rights of reproduction in any form reserved
248
PERLMAN,
CHIKARMANE,
posite gels are superior to high-concentration agarose gels in their ability to fractionate small DNA fragments. The composite gels are inexpensive, easily poured from a melt, and have excellent uv and visible light transparency. MATERIALS
AND
METHODS
Electrophoretic conditions. Tris-borate EDTA (TBE)’ buffer (9) containing ethidium bromide (1 &ml) was used as the electrophoresis buffer in all experiments. Minigels were poured on 75 X 50-mm glass slides which held 10 ml of gel. Samples were loaded in 5% glycerol containing 0.05% each of xylene cyanole FF and bromphenol blue as marker dyes and electrophoresed at 5 V/cm. Preparation of soluble cellulose solution. HEC was prepared as a 2% (w/v) stock solution in distilled water by stirring continuously until the solution became clear and very viscous, after which it was left overnight to ensure homogeneity. NaN3 (0.1%) was added as a bacteriostatic agent. Solution aliquots were weighed rather than pipetted due to high viscosity. Preparation of composite gels. (a) For HEC-, MC-, and GM-agarose gels, hot aqueous agarose was mixed with appropriate volumes of hot polysaccharide solution and TBE buffer. Gels were poured after bubbles had floated to the surface. (b) Amylose, Fico11 400, and dextran-agarose gels were prepared by dissolving the appropriate powder directly into hot agarose. Preparation of sectored gels. Sectored minigels were prepared by inserting, for example, two evenly spaced acrylic dividers between the teeth of the sample well-forming comb. The outer sectors were poured first, and after removal of the dividers, the center section was poured. Tube gel electrophoresis. Quartz tubing was used to allow direct uv photography of ’ Abbreviations MC, methylcellulose; borate EDTA.
used: HEC, hydroxyethylcellulos; GM, galactomannan; TBE,
Tris-
AND
HALVORSON
ethidium bromide-stained DNA within the tube. Grade 5 HEC was mixed with 0.1 vol of 10X TBE containing ethidium bromide and drawn into the tube. The bottom was sealed with dialysis tubing and an elastic band. The membrane ensured electrical conductivity without loss of HEC. As an alternative, a rectangular agarose frame (0.7% agarose in TBE) was formed on uv-transparent plastic. This frame contained the sample wells and held soluble polymers used for DNA fractionation. Buffer in the horizontal chamber maintained electrical contact with the agarose but did not flow over it. After electrophoresis, the uv-transparent plate was placed directly on the uv box for photography. HEC viscosities. The viscosities of the various grades of HEC at defined w/v concentrations are grade 1, 7.5-l 50 cp at 5%; grade 2, 150-400 cp at 2%; grade 3, 1500-2500 cp at 2%; grade 4, 4500-6500 cp at 2%; and grade 5, 3400-5000 cp at 1% solution. Chemicals. Agarose (type ME), HEC, and GM (isolated from locust bean gum) were from the FMC Corp. Ficoll 400 was from Pharmacia. Dextran @&, 5-40 X 106) and MC were from Sigma Chemicals. DNA restriction fragment size standards were from New England Biolabs. RESULTS
Molecular Sieving of DNA in Cellulose Solutions After making the preliminary observation that the addition of HEC to agarose gels enhances the resolution of small DNA fragments, we investigated the possibility that HEC itself had intrinsic sieving capability. DNA restriction fragments ranging from approximately 70 to 1400 bp were applied to the tops of quartz tubes holding HEC solutions and were electrophoresed vertically. Grade 5 HEC (having maximum intrinsic viscosity) was used to minimize diffusion-related mixing during solution-phase electrophoresis. DNA fragments were partially resolved in this medium despite some electro-
IMPROVED
ELECTROPHORETIC
phoretic coning artifacts (Fig. 1, gel a). A technically better result was obtained with horizontal electrophoresis in DNA in HEC solution contained within an agarose frame (Fig. 1, gel b). The best resolution (Fig. 1) in this liquid sieve was obtained for DNA fragments smaller than 600 bp (1.5% HEC) or 400 bp (2.0% HEC). Methylcellulose and galactomannan acted as comparable molecular sieves in liquid phase; solutions containing as little as 0.5% (w/v) galactomannan were effective in fractionating DNA fragments smaller than 70 bp (data not shown). These experiments showed that a number of nonionic polysaccharide solutions had intrinsic DNA-sieving ability. However, the practical use of such nongelled sieving matrices is limited due to diffusion and convection artifacts.
Addition of HEC to agarose gels improves the sieving of small DNA fragments. Agarose and HEC were mixed to form composite gels since reduction of diffusion-related artifacts inherent in the liquid HEC sieving medium was desirable. To compare sieving in a composite gel with that in a simple agarose gel having a similar “solids” content, a sectored gel was utilized, with DNA being simulta-
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neously electrophoresed in gel sectors containing either 0.7% agarose with 1.3% HEC (grade 4) or simple 2.0% agarose. The addition of HEC clearly improved the sieving potential of agarose in the separation of small DNA fragments (Fig. 2, gels a and b). Small DNA fragments were better resolved in the HEC-agarose gel (Fig. 2). The break in the HEC-agarose curve around 600 bp is consistent with that observed in Fig. 1, demonstrating the strong influence of HEC on the fractionation behavior of the composite gel. The excellent linearity maintained at the small molecular size end of the HEC-agarose plot (and poor linearity for agarose) suggests that HEC solutions have a smaller “pore size” than conventional agarose gels. Resolution of DNA fragments larger than 1000 bp appeared to be comparable in the two gels (data not shown).
Sieving ejiciency is a function of HEC concentration. If HEC were primarily responsible for DNA sieving in HEC-agarose gels, then varying the agarose concentration at a fixed HEC level should have little effect on DNA fragment mobility. This prediction was confirmed in experiments varying agarose content between 0.4 and 1.6% (w/v)
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FIG. 1. Electrophoresis of DNA in HEC solutions. HueIII-cleaved 4X174 RF DNA (150 ng) was electrophoresed (gel a) in submerged vertical quartz tubes (3-mm id. X 15-cm length) containing (w/v) 1.5% (left) or 2.0% (right) HEC, grade 5, dissolved in buffer containing ethidium bromide (2 pg/ml). Mobilities of the electrophoresed fragments are plotted: 1.5% HEC (a), 2.0% HEC (A). $X174 RF DNA fragments (gel b, left) and Mspkleaved pBR322 DNA (gel b, right) were electrophoresed in a 2% HEC solution (grade 5) contained in a horizontal agarose frame.
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FIG. 2. Electrophoresis of DNA in HEC-agarose and GM-agarose composite gels. A two-part gel was prepared containing 2.0% agarose (a) and 1.3% HEC (grade 4) plus 0.7% agarose (b). The minigel (&cm length) was submerged in buffer and preelectrophoresed for 20 min (5 V/cm) before the sample DNAs were loaded. HueIII-cleaved dX I74 RF DNA (lanes 1 and 3) and MqI-cleaved pBR322 DNA (lanes 2 and 4) (200 ng each) were applied to the gel. Plots of DNA mobility include both &Xl74 and pBR322 restriction fragments: agarose gel (0); HEC-agarose gel (A). Another two-part gel was prepared containing 2.0% agarose (c) and 0.7% agarose plus 0.5% GM (d). DNA markers and electrophoresis were as described above except that lanes 1 and 3 contained a mixture of HaeIII-cleaved @X 174 RF DNA and MS&cleaved pBR322 DNA fragments.
with HEC fixed at 1.0% (data not shown). Subsequently, with agarose concentration held constant at 0.7%, we varied the HEC concentration and monitored DNA sieving ability (Fig. 3A). At the lowest concentration of HEC, 0. l%, there was only minor retardation of small DNA fragments but with increasing HEC levels, DNA fragment mobility steadily decreased relative to fragment migration rates in conventional agarose. The accompanying change in the slopes of the log molecular size vs mobility curves reflects improvement in sieving efficiency. Normalizing these curves to a common migration distance accentuated the difference in slopes (and hence resolution). The changing y-coordinate position of the breakpoint in the curves in Fig. 3A establishes the maximum DNA fragment size which can be optimally fractionated using a given concentration of HEC. This position,
which decreased as the HEC concentration increased, may represent an “exclusion point” at which pore size in the HEC-contaming matrix becomes limiting. Above this point, sieving may more closely resemble the original agarose gel. The upper limit to the concentration of HEC which may be added to agarose gels is determined by solubility [approximately 2% (w/v)]. For 0.5-0.7% (w/v) agarose gels, addition of more than 2-2.5% HEC produced a cloudy granular gel having inferior sieving properties.
Sieving eficiency improves with increasing intrinsic viscosity of HEC. Several observations were made by comparing HEC-agarose composite gels containing HEC of differing viscosities. (i) Gel strength as determined by resistance to gel collapse (for 0.2% agarose1.O% HEC gels subjected to 50,OOOg centrifugation for 30 min) was much greater with high- than with low-viscosity HEC. The per-
IMPROVED
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centage volume collapse for a gel containing grade 1 HEC was 59% vs only 3 and I%, respectively, for HEC grades 3 and 4. Supernatants liberated from these partially collapsed gels contained the original concentration of HEC (l.O%, w/v), suggesting that HEC was rather loosely held within the aga-
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FIG. 3. Effect of increasing HEC concentration and HEC viscosity on DNA sieving. A four-part gel was prepared containing 0.7% agarose with differing concentrations of HEC (viscosity grade 4) in each part. To prevent HEC leaching, the minigel (5cm length) was covered with plastic wrap prior to submerging in buffer. HindIII-cleaved phage X DNA and HueIII-cleaved &Xl74 RF DNA (200 ng each) were applied to the gel. (A) The relative electrophoretic mobilities of DNA fragments are plotted against log of molecular size. sectors of gel contained unsupplemented agarose (0) and agarose plus 0.1% (0), 0.3% (+), and 1.0‘76 (A) (w/v) HEC. (B) A four-part gel was prepared as above except that the gel sectors contained 0.5% agarose plus 1.5% HEC of differing intrinsic viscosities. HueIII-cleaved $X I74 RF DNA (200 ng) was electrophoresed in gel lanes containing HEC viscosity grade 1 (O), grade 2 (0), grade 3 (+), and grade 4 (A).
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rose gel matrix; i.e., the affinity of HEC for water was greater than that for the agarose matrix. (ii) Loss of HEC from submerged gels to the surrounding buffer (termed leaching) was measurable with all HEC viscosities tested. Leaching occurred more rapidly with low- than with high-viscosity HEC and explained why preelectrophoresis was sometimes required to stabilize the electrophoretic properties of submerged HEC-agarose gels. However, HEC leaching was prevented and preelectrophoresis was obviated by covering the gels (except for the sample wells) with a microscope slide or plastic wrap prior to submerging. Given the above observations showing physical properties of agarose gels varying with HEC viscosity, we studied DNA sieving as a function of HEC viscosity. A sectored gel containing 0.5% agarose and 1.5% HEC of varying viscosity (grades 1, 2, 3, and 4) was prepared. The gel was covered with a glass slide, submerged in buffer, loaded with DNA, and electrophoresed. The higher the HEC viscosity, the better the separation of DNA fragments (Fig. 3B). The low-intrinsicviscosity grade 1 HEC did not sufficiently improve the sieving properties of conventional agarose to be considered useful. Based on these experiments adequate intrinsic viscosity is believed to be a prerequisite for effective molecular sieving by soluble polymers. Other Carbohydrate Polymers May Have a Positive, Neutral, or Negative Efect on DNA Fractionation by Agarose Having demonstrated that soluble cellulose derivatives were capable of DNA sieving, we investigated the sieving properties of other nonionic carbohydrate polymers. As examples, four polymers having high molecular weights but differing in physical structure were chosen. GM is a polymer of #?l-4linked mannose residues with single a-D-galactosyl side chains. Ficoll 400 (-400,000 Da), is a synthetic copolymer of sucrose and epichlorohydrin and has a branched struc-
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ture. Dextran (5-40 X 10” Da) is a linear polymer consisting mainly of LY-1,6-linked glucose residues. Soluble starch (amylose) is an cr l-4-linked glucose polymer and has a helical structure. For each polymer, sectored agarose gels were prepared with the sectors containing differing concentrations of the above polymers (between 0 and 4.0%, w/v). Initially, DNA fragment separation was monitored in composite gels containing GM. $X174 RF and pBR322 DNA fragments were electrophoresed in 0.7% agarose containing 0.5% GM and in a simple 2.0% agarose gel (Fig. 2, gels d and c, respectively). The significant contribution to molecular sieving and the increase in resolution produced by GM were especially pronounced for DNA fragments larger than 200 bp (cf. top six restriction fragments, lanes 2 and 4, Fig. 2, gels c and d). Increasing the GM concentration above 0.5% should further increase the resolution of fragments ~200 bp in a manner analogous to HEC (Fig. 3A). However, due to the rubbery consistency and high gelling temperature of GM-agarose solutions, practical use of such higher GM concentrations would probably require partial hydrolysis of either the GM or the agarose. The minimal amount of GM leaching from GM-agarose gels, attributable to strong intermolecular association (lo), obviated measures to prevent leaching which occurred with submerged HEC-agarose gels. With regard to the other polymers, electrophoresis in agarose-containing dextran or Fico11 of DNA fragments ranging in size from 0.10 to 23 kb showed essentially no alteration in DNA migration rates (data not shown). This constancy was maintained despite significant changes in sol viscosity apparent before gel formation and in gel strength and elasticity resulting from the mixed compositions. Amylose-containing agarose gels, however, exhibited a remarkably altered electrophoretic sieving “phenotype.” While DNA fragments larger than approximately 4000 bp migrated at comparable rates in gels with and without starch, smaller fragments were
AND
HALVORSON
significantly retarded by the starch. A great range of DNA fragment sizes can be simultaneously displayed in a single amylose-agarose gel (Fig. 4). This electrophoretic “compression,” which is analogous to that produced by a gradient gel composition, is essentially opposite to the effect produced by HEC (Fig. 3A) and may be useful in surveying or comparing restriction digestion patterns from large DNA segments. We suggest that the selective retardation of small DNA fragments in amylose-agarose represents some type of size-exclusion process. If, in contrast, the retardation were caused by amylose binding to DNA, then we would expect all DNA fragment mobilities to be proportionately affected. DISCUSSION
A number of nonionic soluble polymers have been shown to sieve DNA molecules during electrophoresis. These polymers, which include soluble cellulose derivatives and galactomannan, can replace a substantial proportion of the agarose in conventional electrophoretic gels. Thus, a 1% HEC solution solidified by a minimum but sufficient concentration of agarose, e.g., approximately 0.4%, can replace a 2.0% agarose gel. A comparison of HEC-agarose and conventional agarose-sieving characteristics for small DNA fragments suggests that the effective pore size is smaller in the HEC network than in the agarose matrix. This reduction in pore size leads to increased resolution of DNA fragments as well as to improved linearity in the plot of log base pairs vs mobility, especially in the lower size range (see Fig. 2). Other advantages of HEC-containing gels over conventional agarose gels are (i) improved transparency with both uv and visible light, (ii) lower material cost, and (iii) reduced electrophoretic run time. We have produced rapidly dissolving dry mixtures of agarose and soluble cellulose which will soon be commercially available. Fractionated macromolecules are easily eluted and precipitated from HEC-agarose
IMPROVED
ELECTROPHORETIC
gels for analytical and preparative purposes. For example, HEC remains soluble in aqueous solutions containing >70% ethanol and does not interfere with the precipitation and subsequent enzymatic treatment of DNA restriction fragments. Thus we have recovered DNA restriction fragments having 5’ protruding ends from HEC-agarose gels treated with DNA polymerase Klenow enzyme to produce blunt ends and successfully ligated this DNA with cleaved pBR322 DNA. Plasmid DNA fragments similarly purified have been nick-translated, producing hybridization probes of specific activity equal to that of DNA purified from conventional agarose gels. We have additionally shown that DNA fragments in HEC-agarose gels can be electrophoretically transferred to nylon and nitrocellulose membranes (unpublished results). Besides increasing the sieving ability of agarose gels, cellulose derivatives greatly in-
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crease the viscosity of the aqueous phase within the gel. We believe that this increased viscosity and the reduction in free water volume within the gel (caused by the hydrophilic polymer) contribute to the high-resolution sharp DNA bands observed in these gels. Other polymers including amylose produced similar DNA band “tightening” yet, together with other nonionic polymers such as Ficoll and high-molecular-weight high-viscosity dextran, did not assist in sieving DNA. Simple low-molecular-weight viscositybuilding agents did not appear to help the sieving process either. For example, we have observed that a 20% sucrose solution added to an agarose gel greatly reduced the rate of DNA electrophoresis. However, all of the DNA fragments were proportionately retarded. With regard to HEC-agarose composite gels, we know that agarose is not required for DNA sieving but minimizes physical mixing
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FIG. 4. DNA sieving in amylose-agarose gels. A three-part gel was prepared containing 0.7% agarose with differing concentrations of amylose. The gel was protected against leaching and the marker DNAs were applied as described in Fig. 3A. Data for agarose (0) [gel (a)], agarose plus 2% amylose (0) [gel (b)], and agarose plus 4% amylose (+) are provided. Lanes 1 and 3, &X174 RF DNA; lanes 2 and 4, phage X DNA.
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within the HEC [cf. Fig. 2 (with agarose) and Fig. 1 (without agarose)]. The weakness of physical and chemical interactions between HEC and agarose was suggested by two direct observations: (i) the concentration of HEC material in supematants of centrifuged composite gels was the same as that originally included in the gels, and (ii) the gelling and remelting temperatures for 0.7% agarose in TBE buffer (35 and 85”C, respectively) were not measurably altered by HEC addition. The physical mechanism by which soluble cellulose or galactomannan interacts with DNA to promote sieving is still unknown. It is possible that helical secondary and/or tertiary structures of carbohydrate polymers play a role in the sieving process. This is deduced from the observations that Ficoll and dextran, which both lack defined secondary and tertiary structures, failed to affect DNA sieving in agarose gels. Helical structures would have considerably more rigidity in solution than random polymers in forming a sieving matrix. Do polysaccharide helices form a systematic noncovalent network of molecules in solution? One clue is that the efficient sieving of DNA occurring in highviscosity HEC solutions was lost with lowviscosity HEC. This sieving characteristic did not change with the addition of a heterologous physical matrix, i.e., the agarose gel used to immobilize the HEC solutions. Therefore we believe that an extended network must exist among HEC molecules themselves for sieving to occur. Furthermore, noncovalent helix-helix binding between soluble polysaccharides, leading to gel formation, has already been demonstrated for agarose and galactomannan ( 10) and also xanthan and galactomannan (11). It is possible that an analogous though weaker binding occurs between agarose and HEC based on
AND HALVORSON
our studies on leaching of HEC from agarose. Curiously, both HEC and galactomannan which have 01-4 linkages increase agarose sieving efficiency, while amylose, with (Yl-4 linkages, decreases the efficiency. All of these polymers including agarose itself (12) have helical structures which appear necessary but apparently are not sufficient for polysaccharides to form sieving matrices. ACKNOWLEDGMENTS We acknowledge support from NIH Grant GM 18904 (D.P.) and from International Minerals & Chemicals Corporation, Northbrook, IL (H.C.). We thank the FMC Corp., Marine Colloids Division, for experimental material and for support in publishing this work and Dr. Hillel Levinson for his review of the manuscript.
REFERENCES 1. Cook, R. B. (1982) U.S. Patent No. 4,319,975. 2. Cook, R. B., and Witt, H. J. (1981) U.S. Patent No. 4,290,9 11. 3. &huller, E., Lefevre, M., and Tompe, L. (1972) Clin. Chim. Acta 42, S- 13. 4. Johansson, B. G., and Hjerten, S. (1974) Anal. Biothem. 59,200-2 13. 5. Renn, D. W., and Mueller, G. P. ( 1970) U.S. Patent No. 3,527,7 12. 6. Bode, H.-J. (1977) Anal. Biochem. 83,204-2 10. 7. Peacock, A. C., and Dingman, C. W. (1968) Biochemistry I, 668-674. 8. Tietz, D., Gottlieb, M. H., Fawcett, J. S., and Chrambach, A. (1986) Electrophoresis 7, 217-220. 9. Maniatis, T., Fritsch, E. F., and Sambrook, J. ( 1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 10. Dea, I. C. M., McKinnon, A. A., and Rees, D. A. (1972) J. Mol. Biol. 68, 153-172. 11. Cairns, P., Miles, M. J., and Morris, V. J. (1986) Nature (London) 322,89-90. 12. Amott, S., Fulmer, A., Scott, W. E., Dea, I. C. M., Moorhouse, R., and Rees, D. A. (1974) J. Mol. Biol. 90,269-284.