Capillary Electrophoresis of DNA

Alan Smith1, Robert J. Nelson2

1 Stanford University, Stanford, California, 2 Dakota Scientific, Sioux Falls, South Dakota
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.9
DOI:  10.1002/0471142700.nc1009s13
Online Posting Date:  August, 2003
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Abstract

Capillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Singleā€base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit.

     
 
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Table of Contents

  • Instrumentation
  • Separation Theory
  • Strategic Planning
  • Basic Protocol 1: Separation of Oligonucleotides
  • Basic Protocol 2: Quantitative PCR Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Separation of Oligonucleotides

  Materials
  • ssDNA 100‐R separation kit (Beckman Coulter) including:
    • 60‐cm, 100‐µm‐i.d. coated capillary
    • ssDNA 100‐R separation gel solution
    • Running buffer: Tris‐borate electrophoresis buffer (reconstitute and store up to 30 days at 4°C)
    • Poly(A) 40‐60 sizing standard (dissolve at 100 µg/ml [3 OD 260 units/ml] in water and store indefinitely at −20°C)
  • Dried ssDNA oligonucleotide sample
  • CE instrument (e.g., Beckman Coulter P/ACE 5510 or equivalent; see Table 10.9.1)

Basic Protocol 2: Quantitative PCR Analysis

  Materials
  • LIFluor dsDNA 1000 kit (Beckman Coulter) containing:
    • Gel buffer mixture (containing separating gel and Tris/borate/EDTA buffer)
    • EnhanCE intercalating dye
    • 65‐cm, 100‐µm‐i.d. coated capillary
    • Standard sizing ladder: HaeIII restriction digest of φX‐174 DNA (dissolve at 10 µg/ml in deionized water and store at −20°C)
  • PCR reaction mixes containing amplicon
  • CE instrument with fluorescent detection (e.g., Beckman Coulter P/ACE 5510 or equivalent)
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Figures

  •   FigureFigure 10.9.1 Schematic of a CE instrument configured for DNA separations.
  •   FigureFigure 10.9.2 CE separation of a standard poly(A)40‐60 mixture of synthetic oligonucleotides.
  •   FigureFigure 10.9.3 A sizing ladder from a HaeIII digest of φX‐174 DNA. Fragment size in bp is indicated.
  •   FigureFigure 10.9.4 CE separation of a synthetic 20‐mer oligonucleotide. The coupling failure products (shorter migration times) and incomplete deprotection products (longer migration times) are clearly visible.
  •   FigureFigure 10.9.5 A portion of the chromatographic output from a PE Biosystems model 373A DNA sequencer using dye terminator sequencing chemistry. Although shown in black and white here, the direct output is color‐coded to more clearly illustrate the peaks corresponding to each base in the sequence.

Videos

Literature Cited

   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2003. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Butler, J., McCord, B., Jung, J., and Allen, R. 1994. Rapid analysis of short tandem repeat HUMTH01 by capillary electrophoresis. BioTechniques 10:1062‐1068.
   Cohen, A., Najarian, D., Paulus, A., Guttman, A., Smith, J., and Karger, B. 1988. Rapid separation and purification of oligonucleotides by high‐performance, capillary, gel electrophoresis. Proc. Natl. Acad. Sci. U.S.A. 85:9660‐9663.
   Elbashir, E.M., Harborth, J., Lendeckel, W., Yalkcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21‐nucleotide RNAs mediated RNA interference in cultured mammalian cells. Nature 411:494‐498.
   Fasco, M., Treanor, C., Spivack, S., Figge, H., and Kaminsky, L. 1995. Quantitative RNA‐polymerase chain reaction‐DNA analysis by capillary electrophoresis and laser induced fluorescence. Anal. Biochem. 224:140‐147.
   Freeman, W.M., Walker, S.J., and Vrana, K.E. 1999. Quantitative RT‐PCR: Pitfalls and potential. BioTechniques 26:112‐125.
   Grossman, P.D. 1991. Effect of molecular orientation and entangled polymer additives on the electrophoresis of biopolymers in free solution. Ph.D. Thesis, University of California, Berkeley.
   Guttman, A. and Schwartz, H. 1995. Artifacts related to sample introduction in capillary gel electrophoresis affecting separation performance and quantitation. Anal. Chem. 67:2279‐2283.
   Jarcho, J. 1994. Restriction fragment length polymorphism analysis. In Current Protocols in Human Genetics (N.C. Dracopoli, J.L. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, and D.R. Smith, eds.) pp. 2.7.1‐2.7.15. John Wiley & Sons, New York.
   Piatak, M., Luk, K.‐C., Williams, B., and Lifson, J.D. 1993. Quantitative competitive PCR for accurate quantitation of HIV DNA and RNA species. BioTechniques 14:70‐81.
   Pon, R., Buck, G., Hager, K., Naeve, C., Niece, R., Robertson, M., and Smith, A. 1996. Multifacility survey of oligonucleotide synthesis and an examination of the performance of unpurified primers in automated DNA sequencing. BioTechniques 21:680‐685.
   Ren, J., Ulvik, A., Ueland, P.U., and Refsum, H. 1997. Analysis of single‐strand conformation polymorphism by capillary electrophoresis with laser‐induced fluorescence detection using short‐chain polyacrylamide as sieving medium. Anal. Biochem. 245:79‐84.
   Rossomando, E., White, L., and Ulfelder, K. 1991. Capillary electrophoresis: Separation and quantitation of reverse transcriptase polymerase chain reaction products from polio virus. J. Chromatogr. B. 656:159‐168.
   Ruiz‐Martinez, M., Salas‐Solano, O., Carilho, E., Kotler, L., and Karger, B. 1998. A sample purification method for ruffed and high‐performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocol. Anal. Chem. 70:1516‐1527.
   Sozer, A.C., Kelly, C.M., Demers, D.B. 1998. Molecular analysis of paternity. In Current Protocols in Human Genetics (N.C. Dracopoli, J.L. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, and D.R. Smith, eds.) pp. 14.4.1‐14.4.26. John Wiley & Sons, New York.
   Ulfelder, K., Schwartz, H., Hall, J., and Sunzeri, F. 1992. Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis. Anal. Biochem. 200:260‐267.
   Zhu, H., Clark, S., Benson, S., Rye, H., and Glazer, A. 1994. High sensitivity capillary electrophoresis of double stranded DNA fragments using monomeric and dimeric fluorescent intercalating dyes. Anal. Chem. 66:1941‐1949.
Key Reference
   Ulfelder, K.J. and McCord, B. 1997. Chapter 11. Separation of DNA by capillary electrophoresis. In Handbook of Capillary Electrophoresis, 2nd ed. (J.P., Landers, ed.) pp. 347‐378. CRC Press, Boca Raton, Fla.
  An excellent reference on all aspects of capillary electrophoresis separations. Chapter 11, on DNA, goes into much greater depth than is possible here on the theory of separation, selection of buffers, and selection of gel matrices.
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