Chapter 8
Pulsed-Field Gel Electrophoresis: Protocols
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND
BIRGIT WEISS
lntroduction
Pulsed-field gel electrophoresis (PFGE) techniques in combination with the
cloning of large fragments of DNA into yeast artificial chromosomes
(YACs) have revolutionized physical mapping in molecular genetics (Barlow and Lehrach 1987; Burke et al. 1987). DNA contigs of megabase length
and physical maps of chromosome bands or entire human chromosomes
are now at the stage of feasibility.
Conventional gel separation techniques use a singlepair of electrodes to
generate the electric field and cannot effectively resolve DNA fragments
much larger than 50 kb. PFGE, however, represents a method for separating
high molecular weight DNA in the range from 10 kb up to several Mbp by
introducing new electrode configurations that generate electrical fields in
alternating orientations (Schwartz and Cantor 1984). This periodic switching (pulsing) of fields forces high molecular weight DNA molecules through
the gel in a "zig-zag" path. Basedon the principle that large DNA fragments
reorient more slowly than smaller fragments do, high molecular weight
DNA can be separated by using Iongerpulses and small DNA molecules
by using shorter pulses. Different PFGE systems have been designed and
are used, e.g., OFAGE (orthogonal field-alternating gel electrophoresis
(Schwartz and Cantor 1984), FIGE (field inversion gel electrophoresis (Carle
et al. 1986), CHEF ( contour-clamped homogeneaus electric fields ( Chu et al.
1986), ROFE (rotating field inversion gel electrophoresis (Southern et al.
1987[SV1]), OD-PFGE and 2D-PFGE (one- and two-dimensional pulsedfield gel electrophoresis). Besides its use for analytical purposes for conCorrespondence to Gudrun A. Rappold, Institute ofHuman Genetics, Im Neuenheimer
Feld 328, Heidelberg, 69120, Germany (phone +49-{0)6221-565068; fax +49-{0}6221565332; e-mail gudrun_rappold@krzmail.krz.uni-heidelberg.de}, Karin Ried, Albrecht
Klink, Ercole Rao, Birgit W eiss
Pulsed-Field Gel Electrophoresis: Protocols
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND
BIRGIT WEISS
lntroduction
Pulsed-field gel electrophoresis (PFGE) techniques in combination with the
cloning of large fragments of DNA into yeast artificial chromosomes
(YACs) have revolutionized physical mapping in molecular genetics (Barlow and Lehrach 1987; Burke et al. 1987). DNA contigs of megabase length
and physical maps of chromosome bands or entire human chromosomes
are now at the stage of feasibility.
Conventional gel separation techniques use a singlepair of electrodes to
generate the electric field and cannot effectively resolve DNA fragments
much larger than 50 kb. PFGE, however, represents a method for separating
high molecular weight DNA in the range from 10 kb up to several Mbp by
introducing new electrode configurations that generate electrical fields in
alternating orientations (Schwartz and Cantor 1984). This periodic switching (pulsing) of fields forces high molecular weight DNA molecules through
the gel in a "zig-zag" path. Basedon the principle that large DNA fragments
reorient more slowly than smaller fragments do, high molecular weight
DNA can be separated by using Iongerpulses and small DNA molecules
by using shorter pulses. Different PFGE systems have been designed and
are used, e.g., OFAGE (orthogonal field-alternating gel electrophoresis
(Schwartz and Cantor 1984), FIGE (field inversion gel electrophoresis (Carle
et al. 1986), CHEF ( contour-clamped homogeneaus electric fields ( Chu et al.
1986), ROFE (rotating field inversion gel electrophoresis (Southern et al.
1987[SV1]), OD-PFGE and 2D-PFGE (one- and two-dimensional pulsedfield gel electrophoresis). Besides its use for analytical purposes for conCorrespondence to Gudrun A. Rappold, Institute ofHuman Genetics, Im Neuenheimer
Feld 328, Heidelberg, 69120, Germany (phone +49-{0)6221-565068; fax +49-{0}6221565332; e-mail gudrun_rappold@krzmail.krz.uni-heidelberg.de}, Karin Ried, Albrecht
Klink, Ercole Rao, Birgit W eiss