Pulsed‐field Gel Electrophoresis

Abstract

Pulsed field gel electrophoresis is a technique used to separate large DNA molecules. It is used in a variety of situations including electrophoretic karyotyping, long‐range physical mapping and the analysis of DNA and chromosome structures.

Keywords: chromosome; DNA; karyotype; mapping

Figure 1.

CHEF (contour‐clamped homogeneous electric field) apparatus showing the change in the electric field between (a) one pulse time and (b) the next.

Figure 2.

Preparative pulsed‐field gel showing the areas of the gel that are removed for staining (indicated by dashed lines). M signifies marker tracks and the darkened area represents digested DNA.

Figure 3.

Preparative pulsed‐field gel slice embedded in a high‐percentage agarose gel and maintained in a conventional gel tank. Small arrows indicate the static electric field and the large arrow shows the direction in which the DNA is eluted from the slice and concentrated in the small darkened area beyond the end of the slice.

Figure 4.

Separation of yeast chromosomes and yeast artificial chromosomes (YACs) by pulsed‐field gel electrophoresis. The sizes refer to the YP148 chromosomes. Running conditions were 1% agarose gel in 0.5 × Tris Borate EDTA buffer run at 6 V cm−1 for 30 h at 15°C with a pulse time of 50 s. The YACs are maintained in the yeast strain AB1380 and the additional YAC chromosomes are indicated.

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References

Chu G (1989) Pulsed field electrophoresis in contour‐clamped homogeneous electric fields for the resolution of DNA by size and topology. Electrophoresis 10: 290–295.

Chu G, Vollrath D and Davis RW (1986) Separation of large DNA molecules by contour‐clamped homogeneous electric fields. Science 234: 1582–1585.

Game JC (1992) Pulsed‐field gel analysis of the pattern of DNA double‐strand breaks in the Saccharomyces genome during meiosis. Developmental Genetics 13: 485–497.

Imai T and Olson MV (1990) Second‐generation approach to the construction of yeast artificial‐chromosome libraries. Genomics 8: 297–303.

Li R, Mignot E, Faraco J et al. (1999) Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library. Genomics 58: 9–17.

Link AJ and Olson MV (1991) Physical map of the Saccharomyces cerevisiae genome at 110‐kilobase resolution. Genetics 127: 681–698.

Maule JC (1997) Physical mapping by pulsed‐field gel electrophoresis. In: Boultwood J (ed.) Gene Isolation and Mapping Protocols, pp. 93–121. Totowa, NJ: Humana Press.

Maule JC, Porteous DJ and Brookes AJ (1994) An improved method for recovering intact pulsed field gel purified DNA, of at least 1.6 megabases. Nucleic Acids Research 22: 3245–3246.

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Wincker P, Ravel C, Blaineau C et al. (1996) The Leishmania genome comprises 36 chromosomes conserved across widely divergent human pathogenic species. Nucleic Acids Research 24: 1688–1694.

Further Reading

Bickmore WA and Bird AP (1992) Use of restriction enzymes to detect and isolate genes from mammalian cells. Methods in Enzymology 216: 224–245.

Birren B and Lai E (1993) Pulsed Field Gel Electrophoresis. A Practical Guide. San Diego: Academic Press.

Krawiec S and Riley M (1990) Organization of the bacterial chromosome. Microbiological Reviews 54: 502–539.

Maule JC (1994) Electrophoretic karyotype analysis. Pulsed field gel electrophoresis. In: Gosden JR (ed.) Chromosome Analysis Protocols, pp. 221–252. Totowa, NJ: Humana Press.

Maule JC (1998) Pulsed field gel electrophoresis. Molecular Biotechnology 9: 107–126.

Monaco AP (ed) (1995) Pulsed Field Gel Electrophoresis. A Practical Approach. Oxford: IRL Press.

Monaco AP and Larin Z (1994) YACs, BACs, PACs and MACs: artificial chromosomes as research tools. Trends in Biotechnology 12: 280–286.

Smith DR (1990) Genomic long‐range restriction mapping. Methods 1: 195–203.

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How to Cite close
Maule, John C(Apr 2001) Pulsed‐field Gel Electrophoresis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003107]