Abstract
Two kinds of semi-liquid separation media are frequently used for the separation of DNA by high performance capillary electrophoresis (CE), namely dynamic or entangled free solution polymer sieving matrix, and dilute liquid-gel solutions. Polymer solutions may be defined in three different functional concentrations: dilute, semidilute, and entangled. In dilute solutions, the polymer molecules are physically separate; as the concentration of polymer increases the molecules begin to interact, and at a critical concentration the polymers begin to physically interact to form an entangled physical network (1–3). These media have the ability to act as size selective sieving agents that partition DNA molecules, but since the media are liquid the injection of fresh media and the removal of expended media are facilitated. The media are typically neutral high molecular weight polymers and polymer sugars, which provide a high uniformity of electrophoretic medium if capillary electroosmotic flow (EOF) effects are suppressed. For the purpose of this review, particular attention will be drawn to entangled free solution capillary electrophoresis (ESCE) using Tris-borate-EDTA (TBE) buffers, with glycerol as an additive. For DNA separations, many different ESCE sieving media and dilute liquid-gel solutions have been used (Table 1): polysaccharide polymers such as, hydroxypropyl cellulose (HPC) (2–4), hydroxypropyl methyl cellulose (HPMC) (5–7), methyl cellulose (8,9), hydroxyethyl cellulose (HEC) (3,7,10–15), dextran (16,17), TreviSol (18), semi-liquid agarose (19,20), and galactomannans (1). Nonsaccharide polymers such as linear polyacrylamides and substituted polyacrylamides (3,7,10–26), linear and branched poly(ethylene oxides), and poly vinyl polymers (27) are also employed for a wide variety of separations purposes.
Keywords
- Methyl Cellulose
- Hydroxyethyl Cellulose
- Hydroxypropyl Cellulose
- Heteroduplex Molecule
- High Performance Capillary Electrophoresis
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References
Sunada W. M. and Blanch H. W. (1997) Polymeric separation media for capillary electrophoresis of nucleic acids. Electrophoresis 18, 2243–2254.
Barron A. E., Soane D. S., and Blanch H. W. (1993) Capillary electrophoresis of DNA in uncross-linked polymer solutions. J. Chromatogr. A 652, 3–16.
Barron A.E., Sunada W.M., and Blanch H.W. (1996) The effects of polymer properties on DNA separations by capillary electrophoresis in uncross-linked polymer solutions. Electrophoresis 17, 744–757.
Mitnik L., Salome L., Viovy J.-L., and Heller C. (1995) Systematic study of field and concentration effects in capillary electrophoresis of DNA in polymer solutions. J. Chromatogr. A 710, 309–321.
Cheng J. and Mitchelson K. R. (1994) Glycerol-enhanced separation of DNA fragments in entangled solution capillary electrophoresis. Anal. Chem. 66, 4210–4214.
Fortina P., Cheng J., Shoffner M. A., Surrey S., Hitchcock W. M., Kricka L. J., and Wilding P. (1997) Diagnosis of Duchenne/Becker muscular dystrophy and quantitative identification of carrier status by use of entangled solution capillary electrophoresis. Clin. Chem. 43, 745–751.
Gelfi C., Perego M., Libbra F., and Righetti P. G. (1996) Comparison of behavior of N-substituted acrylamides and celluloses on double-stranded DNA separations by capillary electrophoresis at 25 degrees and 60 degrees C. Electrophoresis 17, 1342–1347.
Nishimura A., Tsuhako M., Miki T., Ogihara T., and Baba Y. (1998) Analysis of a variable number of tandem repeats in a heart disease gene by capillary electrophoresis with laser-induced fluorescence detector. Chem. Pharm. Bull. (Tokyo) 46, 294–297.
McGregor D. A. and Yeung E. S. (1995) Optimization of capillary electrophoretic separation of DNA fragments based on polymer filled capillaries. J. Chromatogr. A. 652, 67–73.
Nathakarnkitkool S., Oefner P. J., Bartsch G., Chin M. A., and Bonn G. K. (1992) High-resolution capillary electrophoretic analysis of DNA in free solution. Electrophoresis 13, 18–31.
Grossman P. D. and Soane D. S. (1991) Capillary electrophoresis of DNA in entangled polymer solutions. J. Chromatogr. A 559, 257–266.
Atha D. H. (1998) Characterization of DNA standards by capillary electrophoresis. Electrophoresis 19, 1428–1435.
Isenberg A. R., Allen R. O., Keys K. M., Smerick J. B., Budowle B., and McCord B. R. (1998) Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection. Electrophoresis 19, 94–100.
Mansfield E. S., Wilson R. B., and Fortina P. (2001) Analysis of short tandem repeat markers by capillary array electrophoresis, in Capillary Electrophoresis of Nucleic Acids, Vol. 2 (Mitchelson K. R. and Cheng J., eds.), Humana Press Totowa, NJ, pp. 151–161.
Issaq H. J., Chan K. C., and Muschik G. M. (1997) The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products. Electrophoresis 18, 1153–1158.
Heller C. (1998) Finding a universal low-viscosity polymer for DNA separation. Electrophoresis 19, 1691–1698.
Katsuragi K., Kitagishi K., Mizuguchi T., Nagashima T., Kinoshita M., and Kumada H. (1997) Method for detection of epsilon-secondary structure in the precore region of human hepatitis B virus DNA using a fluorescence-based polymerase chain reactionsingle-strand-conformation polymorphism technique with capillary electrophoresis. J. Chromatogr. A 781, 307–314.
Siles B. A. and Collier G. B. (1996) The characterization of a new size-sieving polymeric matrix for the separation of DNA fragments using capillary electrophoresis. J. Capillary Electrophor. 3, 313–321.
Palm A. K. (2001) Capillary electrophoresis of DNA fragments with replaceable lowgelling agarose gels, in Capillary Electrophoresis of Nucleic Acids, Vol. 1 (Mitchelson K. R. and Cheng J., eds.), Humana Press Totowa, NJ, pp. 279–290.
Strutz K. and Stellwagen N. C. (1998) Do DNA gel electrophoretic mobilities extrapolate to the free-solution mobility of DNA at zero gel concentration ? Electrophoresis 19, 635–642.
Pariat Y. F., Berka J., Heiger D. N., Schmitt T., Vilenchik M., Cohen A. S., Foret F., and Karger B. L. (1993) Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices. J. Chromatogr. A 652, 57–66.
Hebenbrock K., Williams P. M., and Karger B. L. (1995) Single strand conformational polymorphism using capillary electrophoresis with two-dye laser-induced fluorescence detection. Electrophoresis 16, 1429–1436.
Gelfi C., Simò-Alfonso E., Sebastiano R., Citterio A., and Righetti P. G. (1996) Novel acrylamido monomers with higher hydrophilicity and improved hydrolytic stability: III. DNA separations by capillary electrophoresis in poly(N-acryloylaminopropanol). Electrophoresis 17, 738–743.
Simò-Alfonso E., Gelfi C., Lucisano M., and Righetti P. G. (1996) Performance of a series of novel N-substituted acrylamides in capillary electrophoresis of DNA fragments. J. Chromatogr. A 756, 255–261.
Righetti P. G. (1995) Macroporous gels: facts and misfacts. J. Chromatogr. A 698, 3–17.
Righetti P. G. and Snyder R. S. (1988) Thermally reversible gels in electrophoresis. I: Matrix characterization. Appl. Theor. Electrophor. 1, 53–58.
Braun B., Blanch H. W., and Prausnitz J. M. (1997) Capillary electrophoresis of DNA restriction fragments: effect of polymer properties. Electrophoresis 18, 1994–1997.
Sunada W. M. and Blanch H. W. (1998) Microscopy of DNA in dilute polymer solutions. Biotechnol. Prog. 14, 766–772.
Zittle C. A. (1951) Reaction of borate with substances of biological interest, in Advances in Enzymology, Vol. 12 (Nord F. F., ed.), Interscience Publishers New York, NY, pp. 493–527.
Böeseken J. (1949) The use of boric acid for the determination of the configuration of carbohydrates, in Advances in Carbohydrate Chemistry, Vol. 4 (Pigman W. W. and Wolfrom M. L., eds.), Academic Press San Diego, CA, pp. 189–210.
Smith J. T., Nashabeh W., and El Rassi Z. (1994) Micellar electrokinetic capillary chromatography with in situ charged micelles. Anal. Chem. 66, 1119–1133.
Bergold A., and Scouten W. H. (1983) Borate chromatography, in Solid Phase Biochemistry: Analytical and Synthetic Aspects, Vol. 66 (Scouten W. H., ed.), Wiley New York, NY, pp. 149–187.
Peng X., Bowser M. T., Britz-McKibbin P., Bebault G. M., Morris J. R., and Chen D. D. (1997) Quantitative description of analyte migration behavior based on dynamic complexation in capillary electrophoresis with one or more additives. Electrophoresis 18, 706–716.
Peats S., Nochumson S., and Kirkpatrick F. H. (1986) Effects of borate on agarose gel structure. Biophys. J. 49, 91a.
Jorgenson J. W. and Lukacs K. D. (1981) Free-zone electrophoresis in glass capillaries. Clin. Chem. 27, 1551–1553.
Shaw D. J. (1969) Electrophoresis. Academic Press London.
Plocek J. and Chmelík J. (1997) Separation of disaccharides as their borate complexes by capillary electrophoresis with indirect detection in visible range. Electrophoresis 18, 1148–1152.
Stellwagen N. C. (1992) Agarose gel pore radii are not dependent on the casting buffer. Electrophoresis 13, 601–603.
Stigter D. (1991) Shielding effects of small ions in gel electrophoresis of DNA. Biopolymers 31, 169–176.
Schellman J. A. (1997) Temperature, stability, and the hydrophobic interaction. Biophys. J. 73, 2960–2964.
Schellman J. A. (1990) A simple model for solvation in mixed solvents. Applications to the stabilization and destabilization of macromolecular structures. Biophys. Chem. 37, 121–140.
Stellwagen N. C., Gelfi C., and Righetti P. G. (1997) The free solution mobility of DNA. Biopolymers 42, 687–703.
Cohen A. S., Najarian D., Smith J. A., and Karger B. L. (1988) Rapid separation of DNA restriction fragments using capillary electrophoresis. J. Chromatogr. 458, 323–333.
Katsivela E. (1997) Analysis of transfer RNA and 5S ribosomal RNA, in Analysis of Nucleic Acids by Capillary Electrophoresis, (Heller C., ed), Chromatography CE Book series, (Altria,K., series ed.), Vieweg Press Germany.
Schwartz H. E., Ulfelder K., Sunzeri F. J., Busch M. P., and Brownlee R. G. (1991) Analysis of DNA restriction fragments and polymerase chain reaction products towards detection of the AIDS (HIV-1) virus in blood. J. Chromatogr. 559, 267–283.
Ulfelder K. J., Schwartz H. E., Hall J. M., and Sunzeri F. J. (1992) Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis. Anal. Biochem. 200, 260–267.
Cheng J., Shoffner M. A., Mitchelson K. R., Kricka L. J., and Wilding P. (1996) Analysis of ligase chain reaction (LCR) products amplified in a silicon chip using entangled solution capillary electrophoresis (ESCE). J. Chromatogr. 732, 151–158.
Fortina P., Cheng J., Kricka L. J., Waters L. J., Jacobson S. C., Wilding P., and Ramsey J. M. (2000) DOP-PCR amplification of whole genomic DNA and microchip-based capillary electrophoresis, in Capillary Electrophoresis of Nucleic Acids, Vol. 2 (Mitchelson K. R. and Cheng J., eds.), Humana Press Totowa, NJ, pp. 211–219.
Cheng J., Kasuga T., Mitchelson K. R., Lightly E. R., Watson N. D., Martin W. J., and Atkinson D. (1994) PCR heteroduplex polymorphism analysis by entangled solution capillary electrophoresis (ESCE). J.Chromatogr.A 677, 169–177.
Kasuga T., Cheng J., and Mitchelson K. R. (2001) Magnetic bead-isolated single-strand DNA for SSCP analysis, in Capillary Electrophoresis of Nucleic Acids, Vol. 2 (Mitchelson K. R. and Cheng J., eds.), Humana Press Totowa, NJ, pp. 135–147.
Inazuka M., Wenz H.-M., Sakabe M., Tahira T., and Hayashi K. (1997) A streamlined mutation detection system: multicolor post-PCR fluorescence labeling and single-strand conormational polymorphism analysis by capillary electrophoresis. Genome Res. 7, 1094–1103.
Kukita Y., Tahira T., Sommer S. S., and Hayashi K. (1997) SSCP analysis of long DNA fragments in low pH gel. Hum. Mutat. 10, 400–407.
Oana H., Hammond R. W., Schwinefus J. J., Wang S. C., Doi M., and Morris M. D. (1998) High-speed separation of linear and supercoiled DNA by capillary electrophoresis. Buffer, entangling polymer, and electric field effects. Anal. Chem. 70, 574–579.
Madabhushi R. S., Vainer M., Dolnik V., Enad S., Barker D. L., Harris D. W., and Mansfield E. S. (1997) Versatile low-viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis. Electrophoresis 18, 104–111.
Kasuga T., Cheng J., and Mitchelson K. R. (1995) Metastable single strand DNA conformational polymorphism (mSSCP) analysis results in enhanced polymorphism detection. PCR Meth. Applic. 4, 227–233.
Kuypers A. W., Willems P. M., van der Schans M. J., Linssen P. C, Wessels H. M., de Bruijn C. H., Everaerts F. M., and Mensink E. J. (1993). Detection of point mutations in DNA using capillary electrophoresis in a polymer network. J. Chromatogr. A 621, 149–156.
Arakawa H., Uetanaka K., Maeda M., and Tsuji A. (1994) Analysis of polymerase chain reaction product by capillary electrophoresis and its application to the detection of single base substitution in genes. J. Chromatogr 66, 89–98.
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Mitchelson, K.R., Cheng, J. (2001). Capillary Electrophoresis with Glycerol as an Additive. In: Mitchelson, K.R., Cheng, J. (eds) Capillary Electrophoresis of Nucleic Acids. Methods in Molecular Biology, vol 162. Humana Press. https://doi.org/10.1385/1-59259-055-1:259
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DOI: https://doi.org/10.1385/1-59259-055-1:259
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