DNA electrophoresis

DNA electrophoresis Lloyd M. Smith University of Wisconsin, Madison, USA The electrophoresis of DNA is a central technology in biological and biomedical research. There is tremendous activity in the development of electrophoresis technology, extending the versatility, power, speed, resolution, and sensitivity of this already extraordinary separation tool. This review covers some of the advances that have been made in the past year. Current Opinion in Biotechnology 1993, 4:37-40 Introduction

Speed

Since the first development of electrophoretic separations b y Tiselius [1], the basic technique has b e e n e x p a n d e d and developed until it n o w includes an astonishing range of different approaches. Virtually all biochemical and genetic research relies upon electrophoresis for the separation and purification of proteins and nucleic acids of interest. The analysis of proteins is most c o m m o n l y achieved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), or a combination of the two techniques. Nucleic acid separations are generally performed in acrylamide gels for smaller fragments (up to 1000 bases in length) and agarose gels for larger fragments (up to 50 000 bases). The advent of pulsed-field gel electrophoresis [2] extended the size range of DNA separations to as much as 10 megabases of DNA. Although these methods usually rely u p o n the use of a gel-sieving medium, the technique of capillary electrophoresis [3] has permitted high resolution separations to be performed in many cases without the use of a gel. The use of entangled polymer solutions as a gel substitute [4"'] has also recently proved to be a powerful approach to such separations, as it eliminates m a n y of the laborious steps of gel preparation.

The major factor limiting the speed of electrophoretic separations is heat transfer efficiency. When an electric field is applied across an electrophoretic medium, the medium acts as a resistor and heat is generated according to Joule's Law. In order to have a stable electrophoretic system, a steady state must be reached in which the rate of heat production is equivalent to the rate of heat dissipation. This steady-state temperature cannot b e so high as to cause deleterious effects in the medium (e.g. boiling) or to damage the analyte. The temperature gradient present in the steady state should not be so large as to affect the resolution of the separation adversely. Important factors affecting heat transfer efficiency include the heat transfer m e d i u m in contact with the electrophoresis cell, the nature of the cooling process (e.g. forced or passive convection), and the surface-to-volume ratio.

As powerful as these methods are, their importance continues to spur interest in further improvements in their capabilities. As in any separation method, one would always like to increase the s p e e d and resolution further. The ability to analyze ever smaller amounts of material is also important. The reliance u p o n gels for m a n y separations has led to research into finding improved matrices. Finally, the need for an understanding at a fundamental level of the separation mechanisms continues to be an important and challenging problem, both in its own right as well as because of its utility in optimizing the technology. In this review, a variety of the advances that have occurred in this field over the last year will be described, falling roughly into the categories described above.

The ability to obtain m u c h higher speeds in electrophoretic separations b y appropriate manipulation of the heat transfer process was first demonstrated with the technique of capillary electrophoresis [3]. In this m e t h o d electrophoretic separations are performed in very narrow (1-100 microns internal diameter) capillary tubes. The high surface-to-volume ratio of these capillaries greatly increases their heat transfer efficiency, allowing the use of much larger electric fields and concomitantly speeding up the separation process. This approach has b e e n applied in a tremendous variety of situations in the past few years, including the separation of carbohydrates, proteins and peptides, single and double-stranded polynucleotides, as well as a variety of smaller molecules [5,6]. The approach is of particular significance in the area of DNA sequence analysis, for which there is a virtually unlimited demand for greater sequencing capability. A system permitting high s p e e d DNA sequencing in an ultrathin slab gel format has b e e n designed [7"']. This system was designed to have an improved heat transfer efficiency. This work w a s followed up by Kostichka e t

Abbreviations DC~irect current; IEF--isoelectric focusing; PAGE---potyacrylamide gel electrophoresis; SDS--sodium dodecyl sulfate. © Current Biology Ltd ISSN 0958-1669

37

38

Analytical biotechnology al. [8"q who designed and built a high sensitivity, fluorescence detection system permitting the four color DNA sequencing strategy to be used in conjunction with ultrathin gels. This system could analyze 18 sampies at a time, for an instrument through-put of 9300 bases per hour, a 7-fold increase over commercial sequencing instruments. Projected improvements will extend this to 50 templates in parallel, for a through-put of 26 000 bases per hour.

Another approach to parallelism in high speed sequencing was taken by Mathies and coworkers [9"-11"]. This group have shown that parallel arrays of many capillaries can be utilized for high speed separations, using a scanning confocal fluorescence detection system. The through-put of the multiple capillaries and ultrathin slab gel systems are comparable, with the primary differences being in the practical issues of gel preparation, loading, and so on. Kambara et al. (Hitachi) [PI"] have extended parallelism further, in a system that permits fluorescence detection to be performed simultaneously from multiple slab gels in parallel [11"]. Although their work to date does not utilize high field separations, it should be possible in principle to combine high field separations with the multiple slab approach to give yet another order of magnitude increase in through-put. Heller and Tullis [12 °] describe another approach, referred to as microelectrophoresis. These workers performed electrophoresis in short (less than one centimeter) gels, obtaining adequate separations of doublestranded restriction fragments or short oligonucleotides in under two minutes. Although the resolution of the separation is necessarily compromised by the short separation distance, for many significant applications the resolution may prove sufficient nonetheless and the very short analysis time is quite valuable.

Detection sensitivity The sensitivity of the detection system used in electrophoresis is always an important issue. The more sensitive the system, the less material is required for analysis and the more versatile the approach. A variety of detection systems are e m p l o y e d routinely in electrophoresis, notably dye staining, silver staining, and radioisotopic labeling for proteins, and either fluorescence detection or radiolabeling for nucleic acids. With regard to nucleic acids, attention has turned increasingly to non-isot0pic methods of detection, and substantial improvements in the sensitivity of fluorescencebased systems have b e e n achieved. The use of laser excitation of fluorescence, and in some cases, of confocal collection systems, has made the sensitivity of fluorescence systems equal to, or in some cases, greater than, that of the more conventional radioisotopic detection systems. Reported detection limits range from 0.1-10

attomoles of fluorescein, depending upon the particular system employed [13,14,15",16"1.

Gel matrices Although great strides have been taken in increasing the speed, resolution, and sensitivity of electrophoretic analysis systems, the real heart of the separation, the gel-sieving matrix itself, has remained relatively unchanged. This is an area of DNA sequence analysis where efforts to commercialize DNA sequencing gels have proved to be largely unsuccessful. The electrophoretic separation employed in sequencing has extremely high resolution, permitting single-stranded DNA fragments differing in length by only one nucleoside subunit out of a total length of as many as 1000 to be separated [17"]. In order to obtain such high resolution of these relatively short DNA molecules, crosslinked polyacrylamide gels are employed which have an average pore size of the order of 10 nm. It is also necessary to include a denaturant, typically urea, in these gels, in order to prevent the formation of secondary structures in the DNA molecules as they are moving through the gel. In order for good separation performance to be obtained from the gel, it is necessary that no charged groups are present. Charged groups cause two types of problems. First, they can cause 'electro-osomotic flow' in which the electric field in the gel acting on the mobile counterions creates pressure and bulk flow of solvent in the gel. This flow pattern can distort the migration and shape characteristics of the bands. Second, the charged groups can adsorb strongly to the analyte molecules in the gel, causing 'tailing' of the bands and decreased resolution. The requirements for gel neutrality and denaturants compromise the chemical stability of the gels. The gels are generally employed at an alkaline pH of about 8.5, and at this pH both acrylamide and urea hydrolyze spontaneously at appreciable rates, producing ionic products. Thus, the shelf life of the gels is short, and it has proved difficult so far to prepare sequencing gels commercially. Instead, these gel mixtures are routinely prepared b y the investigator on a laboratory benchtop. T w o different approaches to this problem have recently begun to attract interest. First, alternative chemical compositions for the sieving matrices have b e e n described, predominantly in the patent literature [P2",P3"]. In principle, the custom design of such polymers should allow the preparation of a sieving matrix which is stable at alkaline pH, thus permitting the elimination of hydrolytically unstable denaturants from the sequencing gels and thereby facilitating commercialization of pre-poured gels. New methods have also b e e n developed for the preparation of capillary gels, using either standard polyacrylamide chemistry or new matrix chemistries, which circumvent some of the difficulties associated with gel preparation (primarily the formation of 'voids' or bubbles in the gels) [P4",18,19].

DNA electrophoresisSmith 39 A second approach is to eliminate cross-linking in gels [4"]. The major issue here is the danger of decreasing the separation p o w e r or resolution. The advantage, however, is the potential replacement of the solid gel matrix with a pumpable, albeit viscous, liquid matrix. Such a solution could be p u m p e d in and out of the electrophoresis cell automatically, permitting new 'gels' to be p r e p a r e d automatically, and opening up a n u m b e r of possible approaches to instrumenting the separations. In one striking example of the potential of the non-crosslinked matrices, Guszczynski and Chrambach [20"] s h o w e d that it was possible to separate yeast chromosomes, megabases in length, in a direct current (DC) electric field, rather than by the pulsed field methods generally used. One possible explanation for the success of this experiment would be the existence of unusually large, effective pore structures in the noncrosslinked matrices, opening up n e w possibilities for the electrophoresis of very large molecules.

and the public sector. There have b e e n new insights into separation mechanisms, which in turn are driving the further d e v e l o p m e n t of the analysis techniques. As important as electrophoresis has b e e n in the history of biological research, it nonetheless appears to b e a field that is still in its infancy.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest 1.

TISELIUSA: A N e w Apparatus for Electrophoretic Analy s i s o f C o l l o i d a l M i x t u r e s . Trans Faraday Soc 1937, 33:524-531.

2.

SCHWARTZDC, CANTOR CR: Separation o f Yeast Chromosome-sized DNAs b y P u l s e d Field Gradient Gel Electrophoresis. Cell 1984, 37:67-75.

3.

JORGENSONJW, LUKACS KD: Zone O p e n - t u b u l a r G l a s s Capillaries. 53:1298-1302.

Separation mechanism A fundamental issue in electrophoresis is the separation mechanism. Although several variants of electrophoresis are fairly amenable to theoretical analysis, and are therefore fairly well understood, there are m a n y important applications for which the principles underlying the separation are less clear. The gel-sieving mechanism in particular is central to almost all nucleic acid separations, and has thus b e e n the subject of considerable scrutiny. DC agarose gel electrophoresis, pulsed-field gel electrophoresis, and DNA sequencing have attracted the most attention. The basic issue is one of resolution: one wishes to maximize the resolution of the separation, but without sacrificing speed. In order to achieve this, it is most helpful to have an understanding of the factors involved. Resolution is determined by two factors: the width of the bands, and the spacing between the bands. Grossman and coworkers [21"] present a thorough analysis of data obtained in DNA sequencing on a commercial system. Luckey and Smith [22"] recently showed that in DNA sequencing, the band width is determined by four factors: diffusion, injection broadening, thermal broadening, and detector volume. These authors d e v e l o p e d a model that correctly predicted the effect of electric field and polynucleotide length u p o n b a n d spacing in DNA sequencing [23"]. Nishikawa and Kambara [24"] analyzed the effects of gel thickness, electric field strength, and other variables u p o n sequencing performance.

Electrophoresis i n Anal Chem 1981,

GROSSMANPD, SOANE DS: E x p e r i t n e n t a l and Theoretical Studies o f DNA Separations b y C a p i l l a r y Electrophoresis i n E n t a n g l e d P o l y l n e r S o l u t i o n s . Biopolymers 1991, 31:1221-1228. A careful theoretical and experimental analysis of DNA separations in non-crosslinked gel-like matrices. 4. •.

5.

EWINGAG, WALLINGFORD RA, OLEFIROW1CZTiM: C a p i l l a r y

Electrophoresis. Anal Chem 1989, 61:292A-303A. 6.

KARGERBL: H i g h - p e r f o r m a n c e C a p i l l a r y Electrophoresis. Nature 1989, 339:641-642.

7. •.

BRUMLEYRL, SMITH LM: R a p i d DNA S e q u e n c i n g b y Hori z o n t a l U l t r a t h i n G e l E l e c t r o p h o r e s i s . Nucleic Acids Res 1991, 19:4121-4126. A novel horizontal apparatus for the preparation and u s e of uitrathin (< 100p.m) polyacrylamide gels is described. A 15-minute electrophoresis is sufficient to resolve 200-300 bases of DNA in a sequencing analysis. 8. •,

KOSTICI-IKAAJ, MARCHBANKSML, BRUMLEYRL, DROSSMAN H, SMITH LM: H i g h Speed Automated DNA S e q u e n c i n g i n U l t r a t h i n Slab Gels. Biotechnology 1992, 10:78~1. A four wavelength fluorescence detection system employing a cooled charge-coupled device detector is described for u s e in conjunction with the horizontal thin gel system described above. The through-put of the system is 9300 bases of DNA sequence p e r hour. 9.

HUANGXC, QUESADA MA, MATHIES RA:

Capillary Array

Electrophoresis Using Laser-excited Confocal Fluoresc e n c e Detection. Anal Chem 1992, 64:967-972. See [11"]. MATHIESRA, HUANG XC: C a p i l l a r y Array Electrophoresis: A n Approach to High-speed, High-throughput DNA Sequencing. Nature 1992, 359:167-169. See [11"]. 10.

HUANGXC, QUESADA MA, MATHIES RA: DNA Sequencing u s i n g C a p i i l ~ r A r r a y E l e e t r o p h o r e s i s . Anal Chem 1992, 64:2149-2154. Capillary array electrophoresis permits the parallel use of multiple capillaries in DNA s e q u e n c i n g (see also [9",10"]). A sensitive, confocal fluorescence detection system is employed, a n d high through-puts similar to those described in [8"'] are obtained. 11.

Conclusion In this short review it is not possible to do justice to the tremendous activity in the field of electrophoresis. N e w geometries, matrices, and instruments are under active and continued development in both the private

12.

HELLERMJ, TULLIS RI-t: Microelectrophoresis for the o f DNA F r a g m e n t s . Electrophoresis 1992, 13:512-520.

Separation

40

Analytical b i o t e c h n o l o g y Electrophoresis is performed in very short, thin polyacrylamide gels, resulting in rapid (two minute) separations. 13.

14.

LUCKEYJA, DROSSMANH, KOSTICHKAAJ, MEADDA, D'CUNHA J, NORRIS TB, SMITH LM: High Speed DNA Sequencing by Capillary Electrophoresis. Nucleic Acids Res 1990, 18:4417-4421. DROSSMANH, LUCKEYJA, KOSTICHKAAJ, D'CUNHAJ, SMITH LM: High Speed Separations o f DNA Sequencing Reactions b y Capillary Eleetrophoresis. Anal Chem 1990, 62:900-903.

15.

QUESADAMA, RYE HS, G1NGRICHJC, GLAZERAN, MATHIES RA: High-sensitivity DNA Detection w i t h a Laser-excited Confocal Fluorescence Gel Scanner. Biotechniques 1991, 10:616-625. A high-sensitivity confocal scanning system for obtaining fluorescence intensity information over an extended two-dimensional region is described. A variety of applications are suggested. SWERDLOWH, ZHANG JZ, CHEN DY, HARKE HR, GREY R, WU S, DOVlCHI NJ: T h r e e DNA Sequencing Methods Using Capillary Gel Elecrophoresis and Laser-induced Fluorescence. Anal Chem 1991, 63:2835-2841. Sensitivity, speed, resolution, and sequencing performance are compared for three different sequencing methods in capillary gels.

The factors determining the width of the bands obtained in DNA sequencing by polyacrylamide gel electrophoresis are described, and employed to predict band width and resolution quantitatively. LUCKEYJA, SMITHLM: A Model for the Mobility o f Singles t r a n d e d DNA in Acrylamide Gel Electrophoresis. Electrophoresis 1993, in press. A semi-empirically modified Ogston sieving model is developed which correctly predicts the effect of molecular length and electric field upon the mobility of single-stranded DNA fragments in polyacrylamide gel electrophoresis. 23. ..

NISHIKAWAT, KAMBARAH: Analysis of Limiting Factors o f DNA Band Separation b y a DNA Sequencer Using Fluorescence Detection. Electrophoresis 1991, 12:623-631. Factors determining the resolution obtained in DNA sequence analysis are determined. The results are used to predict optimal conditions for a given analysis. 24.

16.

NISHIKAWAT, KAMBARAH: High Resolution Separation of DNA Bands b y Electrophoresis w i t h a Long Gel in a Fluorescence-detection DNA Sequencer. Electrophoresis 1992, 13:495-499. The use of a very long gel with moderate field strengths permits the resolution of DNA fragments greater than 1000 bases in length. 17.

18.

19.

ROCHELEAUMJ, GREY RJ, CHEN DY, HARKEHR, DOVICHI NJ: Formamide Modified Polyacrylamide Gels for DNA Sequencing b y Capillary Gel Electrophoresis. Electrophoresis 1992, 13:484-486. SWERDLOWH, DEW-JAGERKE, BRADYK, GREYR, DOVlCHINJ, GESTELAND R: Stability o f Capillary Gels for Automated Sequencing o f DNA. Electrophoresis 1992, 13:475-483.

GUSZCZYNSKIT, CHRAMBACHA: Electrophoretic Separation o f & 20ombe C h r o m o s o m e s i n Polyacrylamide Solutions Using a Constant Field. Biochem Biophys Res Comm 1991, 179:482-486. The separation of very large DNA molecules by DC electrophoresis in non-crosslinked acrylamide solutions is demonstrated. 20. •.

GROSSMANPD, MENCHENS, HERSHEYD: Quantitative Analysis o f DNA-sequencing Electrophoresis. Genetic Analysis Tech Applic 1992, 9:9-16. DNA sequence data produced in commercial sequencing instruments is carefully analyzed using a combination of Ogston sieving and reptation models. 21.

22. •,

LUCKEYJA, SMITHLM: All Analysis o f Resolution in DNA Sequencing b y Capillary Gel Electrophoresis. J Phys Chem 1993, in press.

Patents •.

of special interest of outstanding interest

P1.

HITACHIKK: Fluorescence Detection Type Electrophoresis Apparatus and Its Supporting Vessel. 12/4/89 89JP090842 24/9/91 US5051162A.. A system in which fluorescence data are obtained in parallel from multiple slab gels is described. The authors assert that either single or multiple dye sequencing approaches can be employed. P2.

AT BIOCHEM 1NC: Electrophoretic Media. 20/3/90 90US496338 31/10/91 WO911444989A. Approaches to the chemical synthesis of novel cross-linked gel matrices are presented.

P3.

APPLIEDBIOSYSTEMS INC: High-viscosity Polymer Matrix and Use i n Separation o f Proteins or Nucleic Acids by Electrophoresis or Isoelectric Focusing. 21/1/90 90US472045 8/8/91 WO9111709A, A new approach to the preparation of capillary gels is described.

P4.

INDIANAUNIVERSITY:Capillary Gels F o r m e d b y Spatially Progressive Polymerization Using Migrating Initiator, 26/10/90 90US603983 14/1/92 US5080771A. A controlled method for producing cross-linked polyacrylamide gels without bubbles or voids is described. The method employs a polymerization initiator which may be electrophoresed into the gel solution in a controlled manner.

LM Smith, Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706, USA.