In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes
Résumé
A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [α-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.
Domaines
Alimentation et Nutrition Génomique, Transcriptomique et Protéomique [q-bio.GN] Interactions cellulaires [q-bio.CB] Systématique, phylogénie et taxonomie Evolution [q-bio.PE] Biotechnologies Santé Ecosystèmes Interactions entre organismes Hépatologie et Gastroentérologie Cardiologie et système cardiovasculaire Bactériologie Biochimie [q-bio.BM] Biologie moléculaire Organisation et fonctions cellulaires [q-bio.SC]
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2000_Nucleic Acids Research_KARD et DDIC_e48.pdf (236.55 Ko)
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