In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes.
A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [alpha-(32)P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resulting from a single restriction of the whole genome and (ii) the DDIC (double digestion of isolated chromosome)-PFGE which is the eukaryotic counterpart of complete/complete 2D-PFGE in bacterial genomics.
Brugère
J F
JF
Equipe de Parasitologie Moléculaire et Cellulaire, UPRES A CNRS 6023, Université Blaise Pascal, 63177 Aubière cedex, France.
Cornillot
E
E
Méténier
G
G
Vivarès
C P
CP
D001345:Autoradiography; D002874:Chromosome Mapping / Q000379:methods*; D018983:DNA Footprinting / Q000379:methods*; D004256:DNA Polymerase I; D004271:DNA, Fungal / Q000378:metabolism / Q000528:radiation effects; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D010761:Phosphorus Radioisotopes; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D010761:Phosphorus Radioisotopes; D004256:DNA Polymerase I