Pulsed‐field Gel Electrophoresis of DNA

Abstract

Conventional electrophoresis is not useful for DNA larger than 50 000 base pairs (bp). For large DNA molecules up to 7 million bp (7 Mb) it is necessary to use pulsed‐field electrophoresis. This protocol describes how the procedure is carried out.

Keywords: DNA; separation; PFGE; FIGE

Figure 1.

A Perspex plug mould used for preparing DNA embedded in agarose and a gel comb for making sample loading slots in the gel to accommodate the DNA agarose plugs.

Figure 2.

Effect of pulse time. Saccharomyces cerevisiae (X2180‐1B) chromosomes and phage lambda (c1857Sam7) oligomers run on a 1.5% agarose gel in 0.5× TAE at 6 V cm−1 and 20°C. The pulse times used were (a) 20 s, (b) 60 s and (c) 100 s. The high resolution, showing polymorphism (possibly due to mitotic recombination) in chromosome 3, is clearly visible in the 20 s pulse. In the 100 s pulse, chromosome 12 polymorphism resolves as a doublet near the top of the gel. The general band broadening seen with this pulse time can be reduced by reducing the voltage gradient.

Figure 3.

Effect of agarose gel concentration. A composite gel with agarose concentrations of (a) 1.5%, (b) 1.25%, (c) 1.0% and (d) 0.75% was run in 0.5× TAE at 20°C. The voltage gradient was 4 V cm−1 with a total run time of 35 h and a pulse duration of 90 s. The samples were phage lambda (c1857Sam7) oligomers and Saccharomyces cerevisiae (X2180‐1B).

Figure 4.

Separation of very large DNA. Saccharomyces cerevisiae YNN318, X2180‐1B and Schizosaccharomyces pombe 972 chromosomes run on 0.7% agarose ((a) and (c)) and 0.6% agarose gels ((b) and (d)). Electrophoresis was in 0.5× TAE at 5°C and a voltage gradient of 1.2 V cm−1 was used with 60 min pulses. The total run time was 7 days ((a) and (b)) and 14 days ((c) and (d)). Chromosome 12 of YNN318 is longer than that of X2180‐1B and is separating as such. The rest of the chromosomes of these two strains are of almost the same size, but the X2180‐1B DNA is considerably retarded, clearly demonstrating the drag effects caused by excessive DNA loading in these gels.

Figure 5.

Effect of DNA loading. On the left is an ethidium bromide‐stained gel photograph of four tracks of SfiI‐digested human female (X:21 translocation) DNA. On the right is the corresponding autoradiograph of these same tracks probed with a human X‐chromosome probe that hybridizes to four fragments of 565 kb, 690 kb, 715 kb and 840 kb. The DNA loadings in tracks 1 to 4 were 1.5 μg, 3.0 μg, 3.0 μg and 6.0 μg.

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References

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Carle GF, Frank M and Olson MV (1986) Electrophoretic separation of large DNA molecules by periodic inversion of the electric field. Science 232: 65.

Gardiner K, Lass W and Patterson D (1986) Fractionation of large mammalian DNA restriction fragments using vertical pulsed‐field gradient gel electrophoresis. Somatic Cell and Molecular Genetics 12: 185.

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Further Reading

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

Schwartz DC and Cantor CR (1984) Separation of yeast chromosome‐sized DNAs by pulsed field gradient gel electrophoresis. Cell 37: 67.

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How to Cite close
Southern, Edwin M(Mar 2003) Pulsed‐field Gel Electrophoresis of DNA. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003782]