896 ICGC NORTH AMERICA VOLUME 22 NUMBER 9 SEPTEMBER ZOtM www. chromatographyonline.com The Evolution of Capillary Gel Electrophoresis: From Proteins to DNA Sequencing lectrophoresis (from the Greek induced fluorescence and electrochemical Chromatography words elektron: electron; and detection opened new, previously incon- E phoresis: carrying) is a separation ceivable detection limits as low as in the method in which charged particles migrate zeptomole (10"-^' mol) range. As capillary under the influence of an electric field at gel electrophoresis separates complex sam- various rates, depending upon their charge- ple mixtures in just minutes with excellent to-mass ratios. Electrophoresis as an analyt- teproducibility, even in multicapillary for- ical method was Introduced by Arnie mat, it is becoming an important separa- Tiselius in the 1930s to separate human tion and characterization tool in the Guest Author serum into albumin, a-globulin, (3-glabu- genomics and ptoteomics era. Andras Guttman lin, and 7-^globuIin (1). He demonstrated the potential of electrophotesis as a research The Most Popular Gel: This month's "Milestones tool In his pioneering work for which he Polyacrylamide was awarded the Nobel Prize in 1948. in Chromatography" The basic difference between traditional Latet, antlconvective media such as srarch, slah gel elect rophoresis and capillary gel surveys the evolution of agarose, and polyacrylamide gels were electrophoresis is the utilization of narrow- capillary electrophoresis introduced to eliminate convective trans- bore, fused-silica columns containing the port and reduce diffusion, which resulted and its application in a sieving medium. Capillaries were first filled in the various components of the analyte with cross-linked gels similar to those used wide range of mixture migrating in sharp zones during in conventional polyacryiamide gel elec- biochemical applications. electrophoresis separation (2). Gels also trophoresis and have proven to be particu- acted as molecular sieves for size-based larly efficient in separating protein mole- separations. cules (6). The molecular sieve created by the three-dimensional (3-D) structure of Getting Smaller the polymer chains enabled proper size sep- In the 1980s, a novel, automated, and aration of hiopolymers. Cross-linked gels high-petformance electric-field-mediated are sensitive to temperature, pH, and saddle differential migration technique called cap- voltage changes because of the so-called illary electrophoresis was introduced in "phase transition" phenomena (7). For sta- almost every aspect of basic and applied bilization, the cross-linked gels are attached biomedical and clinical research (3). As an covalendy to the inner capillary surface. instrumental approach to electrk- Samples can he introduced into the capil- field-mediated separations, capillary elec- laries filled with cross-linked gel only by trophoresis offered full automatiog and on- electrokinetic injection, a method that usu- line detection. Using narrow-^bore ally results in sharp peaks due to sample fused-silica capillaries filled with the appro- preconcentration. However, it does not priate polymer sieving media, exceptionally accommodate sample injection from solu- high resolving power was achieved in the tions with high salt concentration. Cross- separation of biologically important macro- linked gels have been used primarily for molecules (4). High separation voltages separating shorter single-stranded DNA ensured rapid separations with excellent molecules and also for the separation of efficiencies — that is, theoretical plate proteins by capillary sodium dodecyl suifate numbers as high as 30 million (5). It gel electro phoresis. The first report on sin- should be noted that the detector trace in gle nucleotide resolution separation of an capillary etectrophoresis is referred to as an oligonucleorides ladder containing 40-60 electropherogram, similar to the term "chro- bases of adenylic acid (8) opened up new Leslie S. Ettre matogram" used in chromatography. High' horizons in DNA sequencing based on Milestones in Chromatography sensitivity detection systems such as laser- modern capillary gel electrophoresis and Editor 898 tCGC NORTH AMERICA VOLUMES NUMBERS SEPTEMBER 2004 www. chromatograp hyo nline.com Low-viscosity polysaccharide matrices have been characterized in capillary elecrrophote- sis for the separation of DNA fragments and have exhibited good separation ability for larger DNA fragments. In the early 1990s, Schwartz and col- leagues attempted to use derivatized cellu- loses as sieving matrices to separate DNA fragments in capillary columns (11). Stan- dard proteins ranging from 14 kDa to 20 22 24 97 kDa were analyzed on hydroxypropyl- Time (min} cellulose in less than 30 min with no appar- ent influence of various buffer composi- Figure 1: Capillary gel electrophoresis separation of single-stranded oligonucleotides ladder tions on the sieving performance (12). The (poiyadenylic acid 40-60) on a cross-linked polyacrylamide gel. (Author's collection.) migration of dsDNA molecules was investi- gated in semidilute hydroxyethylcellulose actually made possible the faster-than-antic- depicts the separation of a DNA restriction solution, revealing the segmental DNA ipated completion of the human genome fragment mixture in a linear polyacry- motion and allowmg quantitative descrip- project. Figure 1 shows an example of such lamide sieving matrix. In some instances, tion of the changing shape of the DNA as separation. sequence-dependent migration behavior of it interacted with the sieving polymer (13). Non-cross-linked, linear polymer matri- dsDNA fragments was observed In capillary Others reported that axial difftision did not ces are not attached ro the inside wall of electrophoresis. Linear polyacrylamide gels account for the total observed band vari- the capillary and feature very flexible, were employed successfully for the separa- ance (14). dynamic pore structures. Actually, the pore tion of standard proteins and other samples Recently, sugar-bearing polyacrylamide sizes of these matrices are defined by of biological origin. copolymers were synthesized and adapted dynamic interactions between the polymer as sieving matrices and capillary coatings chains and can be varied at any time by Sugar-Based Sieving Polymers fbt DNA electrophoresis. Poly(A'.A^- changing such variables as capillary temper- Agarose gel-filled capillaties were exten- dimethylacrylamide) copoiymerized with ature, separation voltage, salt concentration, sively studied for the separation of dsDNA hydrophilic monomers also was used to or pH. Linear polymer gels are not heat fragments (10). In capillary electrophoresis, improve separation performance (15). A sensitive and support simple replacement of a low-concentration (< 0.25%) solution of copolymer of acrylamide and p-D-glucopy- the separation matrix in the capillary by the natural polysaccharide matrices can be ranoside was used as a low-viscosity and pressure. Such gels permit both electroki- applied as a sieving additive for the separa- high-capacity sieving matrix for the separa- netic and pressure injection, enabling large- tion of a range of DNA fragments contain- tion of dsDNA molecules. The growth of scale, routine sample processing. Linear ing as many as 1400 base pairs. The effect the polymer chains was controlled by the polyacrylamide gels were introduced first of temperature and the viscosity of the different reactivity of the two monomers. into coated capillary columns and applied polysaccharide-based sieving medium on The chain length was inversely proportional to the separation of double-stranded DNA the elcctrophoretic behavior of sodium to the number of glucose residues incorpo- (dsDNA) fragments containing as many as dodecyl sulfate (SDS) protein complexes in rated into the copolymer. several thousand base pairs (9). Figure 2 capillary electrophoresis also was studied. Other Sieving Media In addition to the most frequently used cross-linked and linear polyacrylamides and 1 their derivatives (for example, polydimethy- lacryamide), other hydrophilic polymers 10 such as polyethylene oxide, polyvinylpyrrolidone, polyethylene glycol, hydroxyethylcellulose and various cellulose 9 1 derivatives, polysaccharides, and agarose proved useful for the size separation of biopoiymers in natrow-bore capillaries (16). 117 Both polyethylene oxide and 3 poiyvinylpytrolidone matrices provided 2 good separation performance in DNA analysis based on capillary electrophoresis, also featuring self-coating properties (17). 1 : 1 1 10 15 Polyvinylpyrrolidone sieving and wall-coat- Time mid) ing matrix proved useful for rapid molecu- Figure 2: Separation of the DNA restriction fragments by linear polyacrylamide gel. (Author's lar diagnostics capable of detecting com- collection.) Peaks: 1 = 72, 2 = 118, 3 - 194, 4 = 234, 5 = 274, 6 = 281, 7 = 310, 8 = 603, 9 = mon mutations using primer extension 872, 10 = 1078, 11 = 1353 base pairs. technique combined with capillary 900 ICGC NOnrH AMERICA VOLUME 2! NUMBER 9 SEPTEMBEfi 2004 www. chromatogcap hyo nline.com lack of adequate stability of cross-linked lengths of as long as 800 bases were polyacrylamide gels within microbore attained in less than 1 h using polymers columns initiated a rapid development to such as these. find novel, more capillary-friendly sieving matrices. Several reports demonstrated the Getting into Biotech usefulness of non-cross-linked polymeric Capillary SDS gel electrophoresis is a rapid 39 40