Chapter 7
lntroduction to Pulsed-Field Gel Electrophoresis (PFGE)
FENG QIAN AND GREGORY G. GERMINO
Principle
An agarose gel is made up of pores of various sizes with net pore size varying
with concentration of the matrix. The mobility of a DNA molecule depends
on how easily it can pass through these pores. Larger molecules must change
their conformation to get through the smaller pores and thus migrate more
slowly. DNA molecules larger than a certain size must squeeze to get
through even the largest pores, and molecules exceeding this size all migrate
at about the same rate. For ordinary agarose gels run in a uni-directional
electric field, limiting mobility occurs between 20 kb and 40 kb. The significance of this Iimitation becomes clear when it is realized that a single genetic
locus may occupy up to 1 Mb of DNA and that DNA molecules in the individual chromosomes oflower eukaryotes may be 7 Mb or more in length.
However, Schwartz and Cantor (1984) showed that it was possible to resolve
much larger fragments by varying the orientation of the electric field in the
gel during the run. Each time the direction of the electric field is switched,
the molecule is forced to change to a new conformation that will allow it to
move in the new direction. The time required for this reorientation is very
sensitive to molecular weight: larger molecules take more time to realign
themselves than do smaller ones.
Feng Qian, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA, Correspondence to Gregory G.
Germino, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA (phone +0 1-41 0-614-1650; fax
+01-410-614-5129; e-mail ggermino@welch.jhu.edu)
lntroduction to Pulsed-Field Gel Electrophoresis (PFGE)
FENG QIAN AND GREGORY G. GERMINO
Principle
An agarose gel is made up of pores of various sizes with net pore size varying
with concentration of the matrix. The mobility of a DNA molecule depends
on how easily it can pass through these pores. Larger molecules must change
their conformation to get through the smaller pores and thus migrate more
slowly. DNA molecules larger than a certain size must squeeze to get
through even the largest pores, and molecules exceeding this size all migrate
at about the same rate. For ordinary agarose gels run in a uni-directional
electric field, limiting mobility occurs between 20 kb and 40 kb. The significance of this Iimitation becomes clear when it is realized that a single genetic
locus may occupy up to 1 Mb of DNA and that DNA molecules in the individual chromosomes oflower eukaryotes may be 7 Mb or more in length.
However, Schwartz and Cantor (1984) showed that it was possible to resolve
much larger fragments by varying the orientation of the electric field in the
gel during the run. Each time the direction of the electric field is switched,
the molecule is forced to change to a new conformation that will allow it to
move in the new direction. The time required for this reorientation is very
sensitive to molecular weight: larger molecules take more time to realign
themselves than do smaller ones.
Feng Qian, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA, Correspondence to Gregory G.
Germino, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA (phone +0 1-41 0-614-1650; fax
+01-410-614-5129; e-mail ggermino@welch.jhu.edu)